Prame peptide analogues

ABSTRACT

Some embodiments relate to analogs of peptides corresponding to class I MHC-restricted T cell epitopes and methods for their generation. These analogs can contain amino acid substitutions at residues that directly interact with MHC molecules, and can confer improved, modified or useful immunologic properties. Additionally, classes of analogs, in which the various substitutions comprise the non-standard residues norleucine and/or norvaline, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the filing date of U.S.Provisional Patent Application Ser. No. 60/691,889, filed on Jun. 17,2005, the entire text of which is incorporated herein by referencewithout disclaimer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

In certain embodiments, the invention disclosed herein relates toanalogs of peptides corresponding to class I MHC-restricted T cellepitopes and methods for their generation. These analogs can containamino acid substitutions at residues that directly interact with MHCmolecules and can confer improved, modified or useful immunologicproperties. In particular, epitope analogs from the tumor-associatedantigens SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase, and melan-A areidentified. Additionally, classes of analogs, in which the varioussubstitutions comprise the non-standard residues norleucine and/ornorvaline, are disclosed.

2. Description of the Related Art

The Major Histocompatibility Complex and T Cell Target Recognition

T lymphocytes (T cells) are antigen-specific immune cells that functionin response to specific antigen signals. B lymphocytes and theantibodies they produce are also antigen-specific entities. However,unlike B lymphocytes, T cells do not respond to antigens in a free orsoluble form. For a T cell to respond to an antigen, it requires theantigen to be bound to a presenting complex comprised of majorhistocompatibility complex (MHC) proteins.

MHC proteins provide the means by which T cells differentiate native or“self” cells from foreign cells. MHC molecules are a category of immunereceptors that present potential peptide epitopes to be monitoredsubsequently by the T cells. There are two types of MHC, class I MHC andclass II MHC. CD4⁺ T cells interact with class II MHC proteins andpredominately have a helper phenotype while CD8⁺ T cells interact withclass I MHC proteins and predominately have a cytolytic phenotype, buteach of them can also exhibit regulatory, particularly suppressive,function. Both MHC class I and II are transmembrane proteins with amajority of their structure on the external surface of the cell.Additionally, both classes of MHC have a peptide binding cleft on theirexternal portions. It is in this cleft that small fragments of proteins,native or foreign, are bound and presented to the extracellularenvironment.

Cells called antigen presenting cells (APCs) display antigens to T cellsusing the MHC. T cells can recognize an antigen, if it is presented onthe MHC. This requirement is called MHC restriction. If an antigen isnot displayed by a recognizable MHC, the T cell will not recognize andact on the antigen signal. T cells specific for the peptide bound to arecognizable MHC bind to these MHC-peptide complexes and proceed to thenext stages of the immune response.

SUMMARY OF THE INVENTION

SSX-2₄₁₋₄₉ Analog Embodiments

Embodiments include analogs of the MHC class I-restricted T cell epitopeSSX-2₄₁₋₄₉, KASEKIFYV (SEQ ID NO. 1), polypeptides comprising theseanalogs that can be processed by pAPC to present the epitope analogs,and nucleic acids that express the analogs. The analogs can have similaror improved immunological properties compared to the wild-type epitope.

One particular embodiment relates to an isolated SSX-2 peptide having asequence comprising one or more amino acid substitutions of the sequenceKASEKIFYV (SEQ ID NO:1), in an amount sufficient to elicit cytokineproduction from a T cell line generated by immunization against anepitope with the sequence KASEKIFYV (SEQ ID NO:1). In one aspect, theamount sufficient is less than 10 μM. In a further aspect, the amount isless than 3 μM. In yet a further aspect, the amount is less than 1 μM.In one aspect, the one or more amino acid substitutions can include atleast one standard amino acid substitution. “Standard amino acid” asused herein includes any of the 20 genetically encoded amino acids.Thus, in one aspect, the at least one standard amino acid substitutioncan, for example Tyr, Val, Leu, Ala, Ile, Met, Trp, Phe, Asp, Asn, orSer. In a further aspect, the one or more amino acid substitution caninclude at least one non-standard amino acid substitution. Non-standardamino acids include, for example, but not limited to, any of thefollowing: norleucine (Nle), norvaline (Nva), phenylglycine (Phg),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO₂)),α-aminobutyric acid (Abu), α-aminoisobutyric acid (Aib), methyl-leucine(MeLeu), methylvaline (MeVal), β-(3-benzothienyl)-alanine(β-(3-benzothienyl)Ala), O-methyltyorosine (O-methyl-Tyr),cyclohexylalanine (Cha), β-(1-napthyl)-alanine (Nal-1),β-(2-napthyl)-alanine (Nal-2), D-stereoisomer of a standard amino acid,or amino acids wherein the carboxy terminus has been modified to theamide (indicated by —NH2). Thus, in one aspect, the at least onenon-standard amino acid substitution can be Nle, Nva, Abu, or aD-stereoisomer of a standard amino acid. In a further aspect, the one ormore amino acid substitution can include a modified terminal amino acid.In one aspect, the modified terminal amino acid can be an amidatedC-terminal amino acid. In a further aspect at least one of thesubstitutions can be the addition of an amino acid, wherein the additionis a C-terminal addition. In a further aspect, the peptide further caninclude the substitution of conserved amino acids at any site, butpreferably at the P3, P5, or P7 sites which are not expressly involvedin any MHC interactions.

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be A, L, V, I, M, D-Ala, Nal-2,Abu, Aib, Nle, or Nva; P3 can be S; P4 can be E, Q, Nle, or Nva; P5 canbe K; P6 can be I, L, V, Nle, or Nva; P7 can be F; P8 can be Y, F,Phe(4-F); and PΩ (P-omega) at P9 can be V, I, A, Nva, MeVal, or Abu. Insome instances, the sequence is not KASEKIFYV (SEQ ID NO. 1).

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be V, L, M, Abu, Nle, or Nva;P3 can be S; P4 can be E, Q, Nle, or Nva; P5 can be K: P6 can be I, L,V, Nle, or Nva; P7 can be F; P8 can be Y, F, Phe(4-F); and PΩ at P9 canbe V, I, A, Nva, MeVal, Abu, or V—NH₂.

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be A, L, V, M, Abu, Nle, orNva; P3 can be S; P4 can be E, Q, Nle, or Nva; P5 can be K; P6 can be I,L, V, Nle, or Nva; P7 can be F; P8 can be Y, F, Phe(4-F); P9 can be V;and PQ at P10 can be I or L.

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be V; P3 can be S; P4 can be E,Q, Nle, or Nva; P5 can be K: P6 can be I, L, V, Nle, or Nva; P7 can beF; P8 can be Y, F, Phe(4-F); P9 can be V; and PΩ at P10 can be I, L, V,or Nle.

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be L; P3 can be S; P4 can be E,Q, Nle, or Nva; P5 can be K: P6 can be I, L, V, Nle, or Nva; P7 can beF; P8 can be Y, F, Phe(4-F); P9 can be V; and PΩ at P10 can be I, L, V,Nle or Nva.

A further embodiment relates to an isolated peptide having the sequence:K{L, V, M, I, D-Ala, D-Val, Nal-2, Aib, Abu, Nle, or Nva}SEKIFYV (SEQ IDNO. 2); or {F, Phg, Y, Phe(4-F), Phe(4-NO₂), O-methyl-Tyr, orP-(3-benzothienyl-Ala}ASEKIFYV (SEQ ID NO. 3); or {Y, F, or W}{V, M, orI}SEKIFYV (SEQ ID NO. 4); or {F or W}LSEKIFYV (SEQ ID NO. 5); or K{A, V,or L}SEKIFYI (SEQ ID NO. 6); or K{L or V}SEKIFYV-NH₂ (SEQ ID NO. 7); orFVSEKIFY{I, A, Nva, Abu, or MeVal} (SEQ ID NO. 8); or FVS{Q, Nle, orNva}KIFYV (SEQ ID NO. 9); or FVSEK{L, V, Nle, or Nva}FYV (SEQ ID NO.10); or FVSEKIF{F or Phe(4-F)}V (SEQ ID NO. 11); or KASEKIFYV{I or L}(SEQ ID NO. 12); or KVSEKIFYV {I, L, V, or Nle} (SEQ ID NO. 13); orKLSEKIFYV {L, V, Nle, or Nva} (SEQ ID NO. 14).

A further embodiment relates to an isolated peptide having the sequence:K{L, V, M, Abu, Nle, or Nva} SEKIFYV (SEQ ID NO. 15); or {F or Phg}ASEKIFYV (SEQ ID NO. 16; or YVSEKIFYV (SEQ ID NO. 17); or F{L, V, orI}SEKIFYV (SEQ ID NO. 18); or W{L or I}SEKIFYV (SEQ ID NO. 19); or K{Vor L}SEKIFYI (SEQ ID NO. 20); or FVSEKIFY{I or Nva} (SEQ ID NO. 21).

A further embodiment relates to an isolated peptide having the sequence:K{V or L}SEKIFYV (SEQ ID NO. 22); or {F or Y}ASEKIFYV (SEQ ID NO. 23);or FVSEKIFYI (SEQ ID NO. 24).

A further embodiment relates to a class I MHC/peptide complex whereinthe peptide has the sequence of any of the peptides in the embodimentsdescribed above and elsewhere herein. In one aspect, the complex can becross-reactive with a T cell receptor (TCR) that recognizes a class IMHC/SSX-2₄₁₋₄₉ complex. In a further aspect, the complex can be anHLA-A2/SSX-2₄₁₋₄₉ complex.

A further embodiment relates to an immunogenic composition that caninclude any of the peptide embodiments described above and elsewhereherein. In one aspect the peptide can have, for example, the sequence:K{L, V, M, Abu, Nle, or Nva}SEKIFYV (SEQ ID NO. 15); or {F or Phg}ASEKIFYV (SEQ ID NO. 16); or YVSEKIFYV (SEQ ID NO. 17); or F{L, V, orI}SEKIFYV (SEQ ID NO. 18); or W{L or I}SEKIFYV (SEQ ID NO. 19); or K{Vor L}SEKIFYI (SEQ ID NO. 20); or FVSEKIFY{I or Nva} (SEQ ID NO. 21), orK{V or L}SEKIFYV (SEQ ID NO. 22); or {F or Y}ASEKIFYV (SEQ ID NO. 23);or FVSEKIFYI (SEQ ID NO. 24).

Some further embodiments relate to analogs of the MHC class I-restrictedT cell epitope NY-ESO-1₁₅₇₋₁₆₅, SLLMWITQC (SEQ ID NO.25), polypeptidesthat include these analogs that can be processed by pAPC to present theepitope analogs, and nucleic acids that express the analogs. The analogscan have similar or improved immunological properties compared to thewild-type epitope.

One embodiment relates to an isolated NY-ESO-1₁₅₇₋₁₆₅ peptide having asequence comprising one or more amino acid substitutions of the sequenceSLLMWITQC (SEQ ID NO: 25), in an amount sufficient to elicit cytokineproduction from a T cell line generated by immunization against anepitope with the sequence SLLMWITQC (SEQ ID NO: 25). For example, in oneaspect, the amount sufficient can be less than 10 μM. In a furtheraspect, the amount can be less than 3 μM. Also, in a further aspect, theamount can be less than 1 μM. In a further aspect, the amount is lessthan 0.3 μM. In one aspect, the one or more amino acid substitution caninclude at least one standard amino acid. In a further aspect, the oneor more amino acid substitution can include at least one non-standardamino acid. In a further aspect, the one or more amino acid substitutioncan include a modified terminal amino acid. In one aspect, the modifiedterminal amino acid can be an amidated C-terminal amino acid. In afurther aspect, at least one of the substitutions can be the addition ofan amino acid, wherein the addition is a C-terminal addition.

One embodiment relates to an isolated peptide having a sequence inwhich:

-   -   P1 is S, F, K, or W;    -   P2 is L, I, V, Nle, or Nva;    -   P3 is L;    -   P4 is M, L, or N;    -   P5 is W;    -   P6 is I, A, L, V, or N;    -   P7 is T;    -   P8 is Q, E, D, or T;    -   PΩ at P9 is C, V, I, L, A, Nva, Nle, V—NH₂, or L-NH₂; and

wherein the sequence is not SLLMWITQ{C, V, I, L, or A} (SEQ ID NO. 26),FVLMWITQA (SEQ ID NO. 27), or FILMWITQ{L or I} (SEQ ID NO. 28).

Another embodiment relates to an isolated peptide having a sequence inwhich:

-   -   P1 is Y;    -   P2 is L, V, I, Nle, or Nva;    -   P3 is L;    -   P4 is M, L, or N;    -   P5 is W;    -   P6 is I, A, L, V, or N;    -   P7 is T;    -   P8 is Q, E, D, or T;    -   PΩ at P9 is V, I, L, Nva, Nle, V—NH₂, or L-NH₂; and

wherein the sequence is not YVLMWITL (SEQ ID NO. 29) or YLLMWIT{I or L}(SEQ ID NO. 30).

A further embodiment relates to an isolated decamer peptide having asequence {S or Y}LLMWITQ{C or V}{L, I or Nle} (SEQ ID NO. 31).

Yet another embodiment relates to an isolated peptide having a sequenceSILMWITQ{C, V, L, or A} (SEQ ID NO. 32), YLLMWITQ{Nva or Nle}(SEQ ID NO.33), F{L or V}LMWITQ{V, L, or I} (SEQ ID NO. 34), Y{I, Nva, orNle}LMWITQV (SEQ ID NO. 35), YLLLWITQV (SEQ ID NO. 36), or TVLMWITQV(SEQ ID NO. 37).

A further embodiment relates to an isolated peptide having a sequence {Sor F}VLMWITQV (SEQ ID NO. 38), SLMWITQNva (SEQ ID NO. 39), orSNvaLMWITQV (SEQ ID NO. 40).

Still another embodiment relates to an isolated peptide having asequence SNvaLMWITQV (SEQ ID NO. 40).

Some embodiments relate to an isolated peptide. The peptide can includeor consist essentially of a sequence in which:

-   -   P0 is X, XX, or XXX, wherein X specifies any amino acid or no        amino acid; and    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is A, L, V, I, M, D-Ala, Nal-2, Abu, Aib, Nle, or Nva; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   PΩ at P9 is V, I, A, Nva, MeVal, Abu, or V—NH₂, or P9 is V, and        PΩ at P10 is I, L, V, Nle or Nva; and    -   PΩ+1 is X, XX, or XXX, wherein X specifies any amino acid or no        amino acid; and wherein the sequence is not KASEKIFYV (SEQ ID        NO. 1);

The isolated peptide can include or consist essentially of the sequence:

-   -   K{L, V, M, I, D-Ala, D-Val, Nal-2, Aib, Abu, Nle, or Nva}SEKIFYV        (SEQ ID NO. 2); or    -   {F, Phg, Y, Phe(4-F), Phe(4-NO₂), O-methyl-Tyr, or        β-(3-benzothienyl-Ala}ASEKIFYV (SEQ ID NO. 3); or    -   {Y, F, or W}{V, M, or I}SEKIFYV (SEQ ID NO. 4); or    -   {F or W}LSEKIFYV (SEQ ID NO. 5); or    -   K{A, V, or L}SEKIFYI (SEQ ID NO. 6); or    -   K{L or V}SEKIFYV-NH₂ (SEQ ID NO. 7); or    -   FVSEKIFY{I, A, Nva, Abu, or MeVal} (SEQ ID NO. 8); or    -   FVS{Q, Nle, or Nva}KIFYV (SEQ ID NO. 9); or    -   FVSEK{L, V, Nle, or Nva}FYV (SEQ ID NO. 10); or    -   FVSEKIF{F or Phe(4-F)}V (SEQ ID NO. 11); or    -   KASEKIFYV{I or L} (SEQ ID NO. 12); or KVSEKIFYV {I, L, V, or        Nle} (SEQ ID NO. 13); or    -   KLSEKIFYV {L, V, Nle, or Nva} (SEQ ID NO. 14).

The isolated peptide can include or consist essentially of the sequence:

-   -   K{L, V, M, Abu, Nle, or Nva} SEKIFYV (SEQ ID NO. 15); or    -   {F or Phg}A SEKIFYV (SEQ ID NO. 16); or    -   YVSEKIFYV (SEQ ID NO. 17); or    -   F{L, V, or I}SEKIFYV (SEQ ID NO. 18); or    -   W{L or I}SEKIFYV (SEQ ID NO. 19); or    -   K{V or L}SEKIFYI (SEQ ID NO. 20); or    -   FVSEKIFY{I or Nva} (SEQ ID NO. 21).

Also, the isolated peptide can include or consist essentially of thesequence:

-   -   K{V or L}SEKIFYV (SEQ ID NO. 22); or    -   {F or Y}ASEKIFYV (SEQ ID NO. 23); or    -   FVSEKIFYI (SEQ ID NO. 24); or

KVSEKIFYV (SEQ ID NO. 41).

Further, the isolated peptide can include or consist essentially of thesequence KVSEKIFYV (SEQ ID NO. 41).

The isolated peptide can have affinity for a class I MHC peptide bindingcleft. The MHC can be, for example, HLA-A2.

Some embodiments relate to a class I MHC/peptide complex wherein thepeptide can have the sequence of any of the peptides described above orelsewhere herein. The class I MHC/peptide complex can be cross-reactivewith a TCR that recognizes a class I MHC/SSX-2₄₁₋₄₉ complex. The class IMHC/peptide complex can be an HLA-A2/SSX-2₄₁₋₄₉ complex.

Other embodiments relate to a polypeptide that includes a peptide asdescribed above and elsewhere herein, embedded within a liberationsequence.

Still further embodiments relate to immunogenic compositions thatinclude a peptide as described above or elsewhere herein.

Other embodiments relate to nucleic acids encoding or nucleic acid meansfor expressing a polypeptide as described above or elsewhere herein.Also, some embodiments relate to immunogenic compositions that includesuch nucleic acids or nucleic acid means.

Some embodiments relate to methods of inducing, maintaining, oramplifying a CTL response. The methods can include intranodaladministration of a composition as described above and elsewhere herein.

Other embodiments relate to methods of entraining a class IMHC-restricted T cell response, which methods can include intranodaladministration a composition as described above or elsewhere herein. Themethods can further include administration of an immunopotentiatingagent.

Further embodiments relate to methods of inducing, maintaining, orentraining a CTL response, which methods can include intranodaladministration of a composition as described above and elsewhere herein.

Some embodiments relate to isolated peptides that include 1 to 3 aminoacid substitutions in the sequence KASEKIFYV (SEQ ID NO. 1) having anaffinity for a class I MHC binding cleft that is similar to or greaterthan the affinity of KASEKIFYV (SEQ ID NO. 1) for said class I MHCbinding cleft. The halftime of dissociation can be similar to or greaterthan the halftime of dissociation of KASEKIFYV (SEQ ID NO. 1) from saidclass I MHC binding cleft. The isolated peptide can be recognized by Tcells with specificity for the peptide KASEKIFYV (SEQ ID NO. 1).

Still further embodiments relate to isolated peptides that include orconsist essentially of a sequence in which:

-   -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is A, L, V, I, M, D-Ala, Nal-2, Abu, Aib, Nle, or Nva; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   PΩ at P9 is V, I, A, Nva, MeVal, or Abu;    -   wherein the sequence is not KASEKIFYV (SEQ ID NO. 1);    -   or    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is V, L, M, Abu, Nle, or Nva; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   PΩ at P9 is V, I, A, Nva, MeVal, Abu, or V—NH₂;    -   or    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is A, L, V, M, Abu, Nle, or Nva; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   P9 is V; and    -   PΩ at P10 is I or L;    -   or    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is V; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   P9 is V; and    -   PΩ at P10 is I, L, V, or Nle;    -   or    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is L; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   P9 is V; and    -   PΩ at P10 is I, L, V, Nle or Nva.

Some embodiments relate isolated peptides that include or consistessentially of a sequence in which:

-   -   P0 is X, XX or XXX, wherein X specifies any amino acid or no        amino acid; and    -   P1 is S, F, K, W or Y; and    -   P2 is L, I, V, Nle, or Nva; and    -   P3 is L; and    -   P4 is M, L, or N; and    -   P5 is W; and    -   P6 is I, A, L, V, or N; and    -   P7 is T; and    -   P8 is Q, E, D, or T; and    -   PΩ at P9 is C, V, I, L, A, Nva, Nle, V—NH₂, or L-NH₂; and    -   PΩ+1 is X, XX, XXX, wherein X specifies any amino acid or no        amino acid; and    -   wherein the sequence is not SLLMWITQ{C, V, I, L, or A} (SEQ ID        NO. 26), FVLMWITQA (SEQ ID NO. 27), FILMWITQ{L or I} (SEQ ID NO.        28), YVLMWITL (SEQ ID NO. 29) or YLLMWIT{I or L} (SEQ ID NO.        30).    -   P1 is S, F, K, or W;    -   P2 is L, I, V, Nle, or Nva;    -   P3 is L;    -   P4 is M, L, or N;    -   P5 is W;    -   P6 is I, A, L, V, or N;    -   P7 is T;    -   P8 is Q, E, D, or T;    -   PΩ at P9 is C, V, I, L, A, Nva, Nle, V—NH₂, or L-NH₂; and    -   wherein the sequence is not SLLMWITQ{C, V, I, L, or A} (SEQ ID        NO. 26), FVLMWITQA (SEQ ID NO. 27), or FILMWITQ{L or I} (SEQ ID        NO. 28);    -   or    -   P1 is Y;    -   P2 is L, V, I, Nle, or Nva;    -   P3 is L;    -   P4 is M, L, or N;    -   P5 is W;    -   P6 is I, A, L, V, or N;    -   P7 is T;    -   P8 is Q, E, D, or T;    -   PΩ at P9 is V, I, L, Nva, Nle, V, V—NH₂, or L-NH₂; and    -   wherein the sequence is not YVLMWITL (SEQ ID NO. 29) or        YLLMWIT{I or L} (SEQ ID NO. 30).

A further embodiment relates to a class I MHC/peptide complex whereinthe peptide can have the sequence of any of the peptides in theembodiments described above or elsewhere herein. In one aspect, thecomplex can be cross-reactive with a TCR that recognizes a class IMHC/NY-ESO-1₁₅₇₋₁₆₅ complex. In a further aspect, the complex can be anHLA-A2/NY-ESO-1₁₅₇₋₁₆₅ complex.

In one aspect of the above embodiments, the peptide can have affinityfor a class I MHC peptide binding cleft, such as HLA-A2.

A further embodiment relates to a polypeptide comprising the peptidesequence of any of the embodiments described herein in association witha liberation sequence.

A further embodiment relates to an immunogenic composition that includesany of the peptide embodiments described herein. In one aspect, thepeptide can have a sequence as set forth herein.

A further embodiment relates to a nucleic acid encoding any of thepeptide embodiments described herein, but preferably those which do nothave non-standard amino acid substitutions. In a further aspect, thenucleic acid can be encoded in a vector.

A further embodiment relates to an immunogenic composition that includesa nucleic acid encoding any of the peptide embodiments disclosed herein.

A further embodiment relates to a method of inducing a CTL responsecomprising intranodally administering of any of the compositions orpeptides of the embodiments disclosed herein. In a further aspect, themethod can allow for maintaining a CTL response. In a further aspect,the method can allow for amplifying a class I MHC-restricted T cellresponse. In a further aspect, the method can allow for entraining aclass I MHC-restricted T cell response. In a further aspect, the methodalso can include administering an immunopotentiating agent.

Some embodiments relate to isolated peptides having a sequencecomprising one to three or four amino acid substitutions in a nativeepitope sequence, wherein a concentration of the peptide required toelicit cytokine production from a T cell line generated by immunizationagainst an epitope with the sequence is not more than a particularconcentration, for example, 10 μM, 1 μM, 0.3 μM, and the like. The oneto three or four amino acid substitutions can include at least onestandard amino acid substitution and/or at least one non-standard aminoacid substitution, and the like. The at least one non-standard aminoacid can be any of those described herein, for example, a D-stereoisomerof a standard amino acid, Nva, or Nle. The one to three or four aminoacid substitutions can include a modified terminal amino acid, and themodified terminal amino acid can be an amidated C-terminal amino acid.One of the substitutions can be the addition of an amino acid, forexample, the addition can be a C-terminal addition.

Other embodiments relate to peptides having an amino acid sequence thatincludes at least one difference from a sequence of a segment of atarget-associated antigen, the segment having known or predictedaffinity for the peptide binding cleft of a MHC protein, wherein the atleast one difference can be a Nle or Nva residue replacing a residue atan MHC-binding motif anchor position in said segment. The anchorposition can be a primary anchor position, for example, P2 or PΩ. Theanchor position can be an auxiliary anchor position. The difference caninclude a Nle or Nva residue replacing a hydrophobic residue in saidsegment. In some aspects I, L, or V can be a preferred residue in theMHC-binding motif anchor position. In some aspects the peptide can havea length of about 8 to about 14 amino acids, or more preferably a lengthof 9 to 10 amino acids, for example.

The MHC protein can be a human MHC protein, for example, class I HLAprotein. The MHC protein can be, for example, a type such as HLA-A2, A3,A24, A30, A66, A68, A69, B7, B8, B15, B27, B35, B37, B38, B39, B40, B48,B51, B52, B53, B 60, B61, B62, B63, B67, B70, B71, B75, B77, C4, Cw1,Cw3, Cw4, Cw6, Cw7, and Cw10. In some aspects, the MHC protein can beHLA-A2 or A24. The MHC can have an anchor residue binding pocket,wherein the pocket is homologous to the B- or F-pocket of HLA-A*0201.The MHC residues responsible for forming binding pockets, and whichbinding pockets accommodate epitope anchor residues and thus define thebinding specificity of the MHC molecule, are well understood in the art.One compilation of such information is found at the FIMM (FunctionalImmunology) website at the hypertext transfer protocol (http://)“sdmc.lit.org.sg:8080/fimm/.” (See also Schönbach C., Koh J. L. Y.,Sheng X., Wong L., and V. Brusic. FIMM, a database of functionalmolecular immunology. Nucleic Acids Research, 2000, Vol. 28, No. 1222-224; Schönbach C., Koh J L, Flower D R, Wong L., and Brusic V. FIMM,a database of functional molecular immunology; update 2002. NucleicAcids Research, 2002, Vol. 30, No. 1 226-229; and Zhang, C. et al., J.Mol. Biol. 281:929-947, 1998; each of which is hereby incorporated byreference in its entirety). Also, the peptide can have at least onebinding characteristic that is substantially the same as, or betterthan, a corresponding characteristic of said segment for said MHC. Forexample, the binding characteristic can be elevated compared with thatof said segment. In some embodiments, the binding characteristic can beaffinity or stability of binding for example.

The peptide can have an immunogenicity that is substantially the sameas, or better than, the immunogenicity of the segment. Theimmunogenicity can be increased. The immunogenicity can evoke an immuneresponse that is cross-reactive to said segment or can evoke a CTLresponse. The immunogenicity can be assessed, for example, using anMHC-tetramer assay, a cytokine assay, a cytotoxicity assay, by measuringan immune response recognizing the peptide, by measuring an immuneresponse recognizing said segment, using an in vitro immunizationssystem, or any other suitable method. The immunization system caninclude human cells. The immunogenicity can be assessed using an in vivoimmunization system, for example, one that includes a transgenic mouse.The peptide can have an at least similar binding characteristic as saidsegment for said MHC. For example, in some aspects what is considered tobe “similar” can be determined based upon the instant disclosure. Insome particular aspects, “similarity” can be based upon, for example,peptide concentration for half-maximal binding, relative affinity,stability (half time of dissociation) and cross-reactivity/functionalavidity. As an example, a peptide can be considered similar if it hasresults or characteristics that are within twofold, even threefold,four, five or 10-fold of the value for the native peptide. For example,for cross-reactivity/functional avidity, a similar result can be onewhere the data are within three and 10-fold of the native peptide. Asanother example, percentage of binding values can be considered similarwhen within 2, 3, 4, 5, 6, 7, 10, 15 or 20% of the native peptide. Insome aspects, ED₅₀ values can be considered similar when within 2- or3-fold of native sequence. Similar halftime of dissociation can be, forexample, within 2- or 3- fold. As still another example, across-reactivity value that is about 2-fold different from wild-type canbe considered similar. These similar values are exemplary only and givenin the context of some aspects of some embodiments. Other “similar”values can be determined based upon the experiments and teachingsherein.

The peptides can be immunologically cross-reactive with the segment.Thus, the cross-reactivity can be assessed by immunizing with thesegment and assaying recognition of the peptide. Alternatively, thecross-reactivity can be assessed by immunizing with the peptide andassaying recognition of the segment.

The peptide as described above and elsewhere herein can be modified toinclude two differences, for example. In some instances, each differenceindependently can include a Nle or Nva residue. In some instances, onedifference can is not a Nle or Nva substitution. Also, the peptide asdescribed above and elsewhere herein can include three or moredifferences.

The target-associated antigen can be a tumor-associated antigen. Thetarget-associated antigen can be a pathogen-associated antigen.

Other embodiments relate to immunogenic composition that include theinstant peptides as described above and elsewhere herein. Furtherembodiments relate to methods of immunization that include administeringsuch compositions to a mammal, for example, administering directly tothe lymphatic system of the mammal.

Still other embodiments relate to methods of making a T cell epitopeanalogue. The methods can include providing an amino acid sequence of asegment of a target-associated antigen, the segment can have known orpredicted affinity for the peptide binding cleft of a MHC protein;changing at least one amino acid of the sequence corresponding to ananchor position of a MHC binding motif to Nle or Nva; and synthesizing apeptide comprising the changed sequence. The synthesis can be, forexample, chemical synthesis or any other synthetic method.

Some embodiments relate to T cell epitope peptide analogues wherein theanalogue differs from a native epitope peptide by replacement of atleast one native residue corresponding to an anchor position of a MHCbinding motif with a Nle or Nva residue.

Some embodiments relate to isolated peptides including or consistingessentially of a sequence in which:

-   -   P0 is X, XX, or XXX, wherein X specifies any amino acid or no        amino acid; and    -   P1 is G, A, S, Abu, or Sar; and    -   P2 is L, M, I, Q, V, Nva, Nle, or Abu; and    -   P3 is P or W; and    -   P4 is S; and    -   P5 is I; and    -   P6 is P; and    -   P7 is V; and    -   P8 is H; and    -   P9 is P, A, L, S, or T; and    -   PΩat P10 is I, L, V, Nva, or Nle; and    -   PΩ+1 is X, XX, or XXX, wherein X specifies any amino acid or no        amino acid; and    -   wherein the sequence is not GLPSIPVHPI (SEQ ID NO. 42).

The isolated peptide can include or consist essentially of the sequence:

-   -   {S, Sar, or Abu}LPSIPVHPI (SEQ ID NO. 43); or    -   G{M or Nle}PSIPVHPI (SEQ ID NO. 44); or    -   G{L, I, Nva, or Nle}WSIPVHPI (SEQ ID NO. 45); or    -   GLWSIPVHP{Nva or V} (SEQ ID NO. 46); or    -   GLPSIPVH{A or S}I (SEQ ID NO. 47); or    -   GLPSIPVHP{V, L, Nva, or Nle} (SEQ ID NO. 48); or    -   G{Nle}PSIPVHP{Nva, or Nle} (SEQ ID NO. 49); or    -   G{Nva}PSIPVHP{Nva} (SEQ ID NO. 50); or    -   G{V, Nva, or Nle}PSIPVHPV (SEQ ID NO. 51); or    -   {Sar or Abu}LPSIPVHP{V or Nva} (SEQ ID NO. 52); or    -   A{V, I, Nva, or Nle}WSIPVHPI (SEQ ID NO. 53); or    -   AVPSIPVHP{V or Nva} (SEQ ID NO. 54); or    -   A{Nva}PSIPVHPV (SEQ ID NO. 55); or    -   ALWSIPVHP{V or Nva} (SEQ ID NO. 56); or    -   GVWSIPVHP{V or Nva} (SEQ ID NO. 57); or    -   G{Nva}WSIPVHPV (SEQ ID NO. 58).

Also, the isolated peptide can include or consist essentially of thesequence:

-   -   {Abu}LPSIPVHPI (SEQ ID NO. 59); or    -   G{V, Nva, or Abu}PSIPVHPI (SEQ ID NO. 60); or    -   GLPSIPVHP{V or Nva} (SEQ ID NO. 61); or    -   GLWSIPVHP{I or Nva} (SEQ ID NO. 62); or    -   G{Nle}PSIPVHP{Nva} (SEQ ID NO. 63); or    -   G{Nle or Nva}PSIPVHPV (SEQ ID NO. 64); or    -   {A or Abu}LPSIPVHP{V or Nva} (SEQ ID NO. 65); or    -   G{Nva}WPSIPVHP{I or V} (SEQ ID NO. 66); or    -   A{Nva or Nle}WSIPVHPI (SEQ ID NO. 67); or    -   A{V or Nva}PSIPVHPV (SEQ ID NO. 68).

In particular, the isolated peptide can include or consist essentiallyof the sequence:

-   -   {Abu}LPSIPVHPI (SEQ ID NO. 59); or    -   GLPSIPVHP{V or Nva} (SEQ ID NO. 61); or    -   GLWSIPVHPI (SEQ ID NO. 69); or    -   G{Nle}PSIPVHP{Nva} (SEQ ID NO. 63).

Preferably, the isolated peptide can include or consist essentially ofthe sequence: GLPIPVHPV (SEQ ID NO. 70).

The peptide can have affinity for a class I MHC peptide binding cleft,and the class I MHC can be, for example, HLA-A2.

Further embodiments relate to class I MHC/peptide complexes wherein thepeptide has the sequence of a peptide as described above and elsewhereherein. The class I MHC/peptide complex can be cross-reactive with a TCRthat recognizes a class I MHC/PSMA₂₈₈₋₂₉₇ complex. The class IMHC/peptide complex can be an HLA-A2/PSMA₂₈₈₋₂₉₇ complex.

Some embodiments relate to polypeptides that include a peptide sequenceas described above and elsewhere herein embedded within a liberationsequence.

Further embodiments relate to immunogenic compositions that include apeptide as described above and elsewhere herein.

Still other embodiments relate to a nucleic acid encoding or a nucleicacid means for expressing a polypeptide as described above and elsewhereherein, as well as immunogenic compositions that include the nucleicacids or nucleic acid means.

Some other embodiments relate to methods of inducing, maintaining, oramplifying a CTL response. The methods can include intranodaladministration of a composition as described above and elsewhere herein.

Also, some methods relate to methods of entraining a class IMHC-restricted T cell response, which methods can include intranodaladministration of a composition as described above and elsewhere herein.The methods can also include administration of an immunopotentiatingagent.

Other embodiments relate to isolated peptides that include 1 to 3substitutions in the sequence GLPSIPVHPI (SEQ ID NO. 42) and which havean affinity for a class I MHC binding cleft that is similar to orgreater than the affinity of GLPSIPVHPI (SEQ ID NO. 42) for the class IMHC binding cleft. The halftime of dissociation can be similar to orgreater than the halftime of dissociation of GLPSIPVHPI (SEQ ID NO. 42)from the class I MHC binding cleft. The isolated peptide can berecognized by T cells with specificity for the peptide GLPSIPVHPI (SEQID NO. 42).

Some embodiments relate to isolated peptides that include or consistessentially of a sequence in which:

-   -   P0 is X, XX, or XXX, wherein X specifies any amino acid or no        amino acid; and    -   P1 is S, K, F, Y, T, Orn, or Hse; and    -   P2 is L, V, M, I, Nva, Nle, or Abu; and    -   P3 is L, Nva, Nle or Abu; and    -   P4 is Q; and    -   P5 is H; and    -   P6 is L, Nva, Nle, or Abu; and    -   P7 is I; and    -   P8 is G, A, S, or Sar; and    -   PΩ at P9 is L, V, I, A, Nle, Nva, Abu, or L-NH₂; and    -   PΩ+1 is X, XX, or XXX, wherein X specifies any amino acid or no        amino acid; and    -   wherein the sequence is not SLLQHLIGL (SEQ ID NO. 71).

The isolated peptide can include or consist essentially of the sequence:

-   -   {K, F, Y, T, Orn, or Hse}LLQHLIGL (SEQ ID NO. 72); or    -   S{V, M, I, Nva, Nle, or Abu}LQHLIGL (SEQ ID NO. 73); or    -   SL{Nva, Nle or Abu}QHLIGL (SEQ ID NO. 74); or    -   SLLQH{Nva, Nle or Abu}IGL (SEQ ID NO. 75); or    -   SLLQHLI{A, S, or Sar}L (SEQ ID NO. 76); or    -   SLLQHLIG{V, I, A, Nle, Nva, Abu, or L-NH₂} (SEQ ID NO. 77); or    -   {F, Y, T, Orn, or Hse} {Nva, Nle, M, or I}LQHLIGL (SEQ ID NO.        78); or    -   S{Nva, Nle, or M}LQHLIG{Nva, Nle, or V} (SEQ ID NO. 79); or    -   {K, F, Y, T, Orn, or Hse}LLQHLIGV (SEQ ID NO. 80); or    -   {F or T}LLQHLIG{Nle} (SEQ ID NO. 81); or    -   {F or T} {Nva or M}LQHLIG{Nle} (SEQ ID NO. 82).

Also, the isolated peptide can include or consist essentially of thesequence:

-   -   {F, Y, T, Orn, or Hse}LLQHLIGL (SEQ ID NO. 83); or    -   S{Nva, Nle, or M}LQHLIGL (SEQ ID NO. 84); or    -   SLLQHLIG{Nle, Nva, or L-NH₂} (SEQ ID NO. 85); or    -   SLLQH{Nva or Abu}IGL (SEQ ID NO. 86); or    -   S{Nva}LQHLIG{Nle} (SEQ ID NO. 87); or {F or T} {L or        Nva}LQHLIG{Nle} (SEQ ID NO. 88).

Further, the isolated peptide can include or consist essentially of thesequence:

-   -   S{L or Nva}LQHLIG{Nle} (SEQ ID NO. 89); or    -   T{Nva}LQHLIG{Nle} (SEQ ID NO. 90).

The isolated can include or consist essentially of the sequenceS{Nva}LQHLIG{Nle} (SEQ ID NO. 87).

The isolated peptide can have affinity for a class I MHC peptide bindingcleft, and for example, the class I MHC can be HLA-A2.

Embodiments relate to class I MHC/peptide complexes wherein the peptidecan have the sequence of a peptide as disclosed above, and elsewhereherein. The class I MHC/peptide complex can be cross-reactive with a TCRthat recognizes a class I MHC/PRAME₄₂₅₋₄₃₃ complex. The class IMHC/peptide complex can be an HLA-A2/PRAME₄₂₅₋₄₃₃ complex.

Other embodiments relate to polypeptides that include a peptide sequenceas described above and described elsewhere herein in association with aliberation sequence.

Further embodiments relate to immunogenic compositions that include apeptide as described above and described elsewhere herein.

Some embodiments relate to a nucleic acid encoding and a nucleic acidmeans for expressing a polypeptide as described above and describedelsewhere herein, as well as immunogenic compositions that include suchnucleic acids and means.

Still other embodiments relate to methods of inducing, maintaining, oramplifying a CTL response, which methods can include intranodaladministration of a composition as described above and describedelsewhere herein. Some embodiments relate to methods of entraining aclass I MHC-restricted T cell response, which can include intranodaladministration of a composition as described above and describedelsewhere herein. In some embodiments, the methods further includeadministration of an immunopotentiating agent.

Some embodiments relate to isolated peptides that include 1 to 3substitutions in the sequence SLLQHLIGL (SEQ ID NO. 71), having anaffinity for a class I MHC binding cleft that is similar to or greaterthan the affinity of SLLQHLIGL (SEQ ID NO. 71) for said class I MHCbinding cleft. The halftime of dissociation can be similar to or greaterthan the halftime of dissociation of SLLQHLIGL (SEQ ID NO. 71) from saidclass I MHC binding cleft. The isolated peptide can be recognized by Tcells with specificity for the peptide SLLQHLIGL (SEQ ID NO. 71).

Further embodiments relate to methods to generate and resultingcompositions representing peptides that are immune active and carryunnatural amino acids at one or multiple MHC anchor residues.

A further embodiment of the present invention relates to the pSEMplasmid and immunogenic peptides expressed by this plasmid correspondingto Melan-A₂₆₋₃₅ and/or tyrosinase₃₆₉₋₃₇₇ epitopes. The pSEM plasmidencodes the Melan-A and tyrosinase epitopes in a manner that allows fortheir expression and presentation by pAPCs. Details of the pSEM plasmidare disclosed in U.S. Patent Application Publication No. 20030228634,which is incorporated herein by reference in its entirety.

In particular embodiments of the invention, there is provided anisolated peptide analogue of an immunogenic peptide expressed by a pSEMplasmid consisting essentially of the sequence: E{A, L, Nva, orNle}AGIGILT{V, Nva, or Nle} (SEQ ID NO. 91); or Y{M, V, Nva, orNle}DGTMSQ{V, Nva, or Nle} (SEQ ID NO. 92); and wherein the sequence isnot E{A or L}AGIGILTV (SEQ ID NO. 93) or YMDGTMSQV (SEQ ID NO. 94). Theisolated peptide analogue of the invention may be selected from thegroup consisting of ELAGIGILTNva (SEQ ID NO. 95), ENvaAGIGILTV (SEQ IDNO. 96), YVDGTMSQNva (SEQ ID NO. 97), YVDGTMSQV (SEQ ID NO. 98) andYMDGTMSQNva (SEQ ID NO. 99).

In other embodiments the isolated peptide analogue is an analogueconsisting essentially of the amino acid sequence ENvaAGIGILTV (SEQ IDNO. 96). In yet other embodiments the isolated peptide analogue is ananalogue consisting essentially of the amino acid sequence YMDGTMSQNva(SEQ ID NO. 97). In further embodiments the peptide has affinity for aclass I MHC peptide binding cleft. The class I MHC is HLA-A2.

Other embodiments of the invention relate to an isolated peptideanalogue comprising one to three substitutions in the sequenceEAAGIGILTV (SEQ ID NO. 100) having an affinity for a class I MHC bindingcleft that is similar to or greater than the affinity of EAAGIGILTV (SEQID NO. 100) for the class I MHC binding cleft. In still otherembodiments, the halftime of dissociation is similar to or greater thanthe halftime of dissociation of EAAGIGILTV (SEQ ID NO. 100) from theclass I MHC binding cleft. In other embodiments, the isolated peptide isrecognized by T cells with specificity for the peptide EAAGIGILTV (SEQID NO. 100).

In yet another embodiment, the invention relates to an isolated peptideanalogue comprising one to three substitutions in the sequence YMDGTMSQV(SEQ ID NO. 94) having an affinity for a class I MHC binding cleft thatis similar to or greater than the affinity of YMDGTMSQV (SEQ ID NO. 94)for the class I MHC binding cleft. In yet other embodiments, thehalftime of dissociation is similar to or greater than the halftime ofdissociation of YMDGTMSQV (SEQ ID NO. 94) from the class I MHC bindingcleft. In other embodiments, the isolated peptide is recognized by Tcells with specificity for the peptide YMDGTMSQV (SEQ ID NO. 94).

Embodiments of the invention also relate to a class I MHC/peptidecomplex wherein the peptide has the sequence of the peptide of: E{A, L,Nva, or Nle}AGIGILT{V, Nva, or Nle} (SEQ ID NO. 91); or Y{M, V, Nva, orNle}DGTMSQ{V, Nva, or Nle} (SEQ ID NO. 92); and wherein the sequence isnot E{A or L}AGIGILTV (SEQ ID NO. 93) or YMDGTMSQV (SEQ ID NO. 94). Inother embodiments, the class I MHC/peptide complex is cross-reactivewith a TCR that recognizes a class I MHC/Melan-A₂₆₋₃₅ complex. The classI MHC/peptide complex of is an HLA-A2/Melan-A₂₆₋₃₅ complex.

In still yet another embodiment, the class I MHC/peptide complex iscross-reactive with a TCR that recognizes a class IMHC/Tyrosinase₃₆₉₋₃₇₇ complex. The class I MHC/peptide complex is anHLA-A2/Tyrosinase₃₆₉₋₃₇₇ complex.

Some embodiments of invention relate to a polypeptide comprising thepeptide sequence of E{A, L, Nva, or Nle}AGIGILT{V, Nva, or Nle} (SEQ IDNO. 91); or Y{M, V, Nva, or Nle}DGTMSQ{V, Nva, or Nle} (SEQ ID NO. 92)embedded within a liberation sequence; wherein the sequence is not E{Aor L}AGIGILTV (SEQ ID NO. 93) or YMDGTMSQV (SEQ ID NO. 94). In yet afurther embodiment, the invention relates to an immunogenic compositioncomprising any of the peptides of E{A, L, Nva, or Nle}AGIGILT{V, Nva, orNle} (SEQ ID NO. 91); or Y{M, V, Nva, or Nle}DGTMSQ{V, Nva, or Nle} (SEQID NO. 92); and wherein the sequence is not E{A or L}AGIGILTV (SEQ IDNO. 93) or YMDGTMSQV (SEQ ID NO. 94). The invention also relates to animmunogenic composition comprising a polypeptide comprising any of thepeptides of E{A, L, Nva, or Nle}AGIGILT{V, Nva, or Nle} (SEQ ID NO. 91);or Y{M, V, Nva, or Nle}DGTMSQ{V, Nva, or Nle} (SEQ ID NO. 92), and to anucleic acid encoding such a polypeptide. Embodiments of inventionfurther relate to an immunogenic composition comprising the nucleicacid.

The immunogenic compositions of the invention may be administered as theentraining portion of an immunization strategy against a cancer such asglioblastoma and melanoma, but is not limited to such. In addition,peptides corresponding to the Melan-A₂₆₋₃₅ and tyrosinase₃₆₉₋₃₇₇epitopes and epitope analogues can be administered as the amplificationportion of the same immunization strategy. In preferred embodiments, thepeptide analogues E{A, L, Nva, or Nle}AGIGILT{V, Nva, or Nle} (SEQ IDNO. 91); or Y{M, V, Nva, or Nle}DGTMSQ{V, Nva, or Nle} (SEQ ID NO. 92)may be utilized in the amplification step. The entrain-and-amplifyprotocol employed in the present invention is as disclosed in greaterdetail in U.S. Patent Publication No. 20050079152, and U.S. ProvisionalPatent Application No. 60/640,402, both entitled METHODS TO ELICIT,ENHANCE AND SUSTAIN IMMUNE RESPONSES AGAINST MHC CLASS I-RESTRICTEDEPITOPES, FOR PROPHYLACTIC OR THERAPEUTIC PURPOSES, each of which isincorporated herein by reference in its entirety.

Thus, in one embodiment, a method of inducing, maintaining, oramplifying a CTL response is provided. The method can include intranodaladministration of an immunogenic composition comprising a nucleic acidencoding a polypeptide comprising the peptide sequence of an immunogenicpeptide expressed by a pSEM plasmid consisting essentially of thesequence: E{A, L, Nva, or Nle}AGIGILT{V, Nva, or Nle} (SEQ ID NO. 91);or Y{M, V, Nva, or Nle}DGTMSQ{V, Nva, or Nle} (SEQ ID NO. 92). Infurther embodiments, there is provided a method of entraining a class IMHC-restricted T cell response comprising intranodal administration ofthe immunogenic composition and an immunopotentiating agent. In yetother embodiments, there is provided a method of inducing, maintaining,or entraining a CTL response comprising intranodal administration of anyimmunogenic composition disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B summarize substitutions for SSX-2₄₁₋₄₉ analogs at eachindividual amino acid position for nonamers and decamers, respectively.

FIG. 2 is a schematic diagram of the methodology of a preferredembodiment for identifying analogs.

FIG. 3 is a table showing the cross-reactivity and functional avidity ofSSX-2₄₁₋₄₉ analogs substituted at a single position.

FIG. 4 is a table showing the cross-reactivity and functional avidity ofSSX-2₄₁₋₄₉ analogs substituted at two positions.

FIG. 5 is a table showing the cross-reactivity and functional avidity ofSSX-2₄₁₋₄₉ analogs substituted at more than two positions.

FIG. 6 is a table showing the cross-reactivity and functional avidity ofSSX-2₄₁₋₄₉ decamer analogs encompassing the nominal 41-49 peptide.

FIG. 7 is a timeline showing the injection schedule of the SSX-2₄₁₋₄₉analogs.

FIG. 8 is a bar graph showing the activity of the SSX-2₄₁₋₄₉ A42V, A42Lanalogs and wild-type in lysis of tumor cells.

FIG. 9 is a timeline showing the injections schedule for in vivocytoxicity studies and ex vivo cytotoxicity studies as well as theSSX-2₄₁₋₄₉ analog peptide used for the boost.

FIG. 10 is a table showing the in vivo specific lysis results for anumber of the analogs as compared to a control (wild-type peptide) andEAA (Melan A 26-35).

FIG. 11 is a table showing the in vivo specific lysis results for anumber of the SSX-2₄₁₋₄₉ analogs as compared to a control (wild-typepeptide) and EAA as well as MHC binding and MHC stability.

FIG. 12 is a bar graph showing the percent specific lysis of tumor cells(624.38 human tumor cells) achieved following immunization with a numberof analogs as compared to a wild-type control.

FIGS. 13A-C are tables summarizing the substitutions at each individualamino acid position for nonamers and decamers, respectively, as well asthe results obtained for each.

FIG. 14 is a timeline showing the injection schedule used for analysisand testing of the NY-ESO-1 analogs.

FIGS. 15A-C show the specific elimination of target cells as measured byremoving the spleens and PBMC from challenged animals and measuring CFSEfluorescence by flow cytometry.

FIGS. 16A and B are bar graphs showing the in vivo cytotoxicity againsttarget cells coated with wild-type peptide after boost with NY-ESO-1analogs.

FIGS. 17A and B are bar graphs showing an ex vivo analysis of theability of the analogs to trigger enhanced immunity against thewild-type epitope as assessed by cytokine production.

FIG. 18 illustrates a protocol for validating the antigenicity of thePSMA₂₈₈₋₂₉₇ epitope, as well as the results of the testing.

FIG. 19 is a table showing the cross-reactivity and functional avidityof PSMA₂₈₈₋₂₉₇ analogs substituted at a single position.

FIG. 20 is a table showing the cross-reactivity and functional avidityof PSMA₂₈₈₋₂₉₇ analogs substituted at two positions.

FIG. 21 is a table showing the cross-reactivity and functional avidityof PSMA₂₈₈₋₂₉₇ analogs substituted at more than two positions.

FIG. 22 is a bar graph showing the immunogenicity of various PSMA₂₈₈₋₂₉₇analogs measured by Elispot.

FIG. 23 is a line graph showing the amplification of anti-PSMA₂₈₈₋₂₉₇response by the I297V analog measured by Elispot.

FIG. 24 is a bar graph showing the results of boosting with the I297Vanalog. The assay showed that the boosting resulted in cytotoxicimmunity against a PSMA⁺ human tumor line.

FIG. 25 illustrates a protocol for validating the antigenicity of thePRAME₄₂₅₋₄₃₃ epitope, as well as the results of the testing.

FIG. 26 is a table showing the cross-reactivity and functional avidityof PRAME₄₂₅₋₄₃₃ analogs substituted at a single position.

FIGS. 27 A and B are tables showing the cross-reactivity and functionalavidity of PRAME₄₂₅₋₄₃₃ analogs substituted at two positions.

FIG. 28 is a table showing the cross-reactivity and functional avidityof PRAME₄₂₅₋₄₃₃ analogs substituted at more than two positions.

FIG. 29 is a bar graph showing the immunogenicity of a PRAME₄₂₅₋₄₃₃analog measured by Elispot.

FIG. 30 shows the results of boosting with the L426Nva L433Nle analog.The assay showed that the boosting resulted in cytotoxic immunityagainst native epitope coated cells.

FIG. 31 shows a protocol for the in vivo evaluation of PRAME analogs, aswell as a bar graph showing the results of the evaluation.

FIG. 32 shows a protocol for the ex vivo stimulation of cytokineproduction in analog induced, native epitope re-stimulated T cells and abar graph showing the results of the evaluation.

FIG. 33 is a bar graph showing the results of boosting with the L426NvaL433Nle analog. The assay showed that the boosting resulted in cytotoxicimmunity against a human tumor cell line.

FIG. 34 depicts a protocol for in vitro immunization to PRAME_(425-433.)

FIG. 35 shows the tetramer analysis results after in vitro immunizationwith PRAME₄₂₅₋₄₃₃ analogs.

FIG. 36 depicts the structure of the plasmid, pCTLR2, a plasmid thatexpresses the PRAME₄₂₅₋₄₃₃ epitope.

FIG. 37 is a bar graph showing the assay results for an experiment inwhich humor tumor cells (624.38) were lysed by T cells primed withplasmid DNA and boosted with peptides.

FIG. 38 is a bar graph showing the tetramer analysis results afterplasmid prime with Tyr₃₆₉₋₃₇₇ and peptide boost with the V377Nva analog.

FIG. 39 is a bar graph showing in vivo response against analoguesTyrosinase and Melan A epitopes.

FIG. 40 is a timeline showing a Tyrosinase analogues immunogenicityevaluation protocol.

FIG. 41 is a bar graph showing the immune response results against624.38 cells contacted with effector cells from HHD primed with plasmidand boosted with Tyr₃₆₉₋₃₇₇ analogs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Peptides encompassing T cell epitopes are usually poor immunogens orimmune modulators due to one of multiple factors: a suboptimalpharmacokinetics profile, limited binding to MHC molecules (reducedK_(on) and increased K_(off)), or decreased intrinsic recognition by Tcells present in the normal immune repertoire (e.g., through variousforms of tolerance). Various strategies have been pursued to improve theimmunologic properties of peptides, particularly the screening and useof peptides in which the sequence differs from the natural epitope. Suchanalogs are known by various names in the art, such as heteroclyticpeptides and altered peptide ligands (APL). The generation of suchanalogs has most often utilized amino acids from the standard set ofgenetically encoded residues (see for example Valmori, D. et al., J.Immunol. 160:1750-1758, 1998). Use of non-standard amino acids hastypically been associated with efforts to improve the biochemicalstability of the peptide (see, for example, Blanchet, J.-S. et al., J.Immunol. 167:5852-5861, 2001).

Generally, analogs can be categorized into the following two mainclasses: (1) modification of peptide anchor residues to achieve betterHLA binding profiles and higher immune responses, and (2) modificationof peptide anchor residues and TCR contact residues to circumvent T celltolerance for self-antigens.

Some embodiments of the invention described herein relate to analogsthat have at least one of the following retained or improved properties,including but not limited to:

1. Cross-reactivity and functional avidity to TCR;

2. Affinity for and stability of binding to MHC class I;

3. In vivo effect on immunity assessed by cytotoxicity;

4. In vivo effect on immunity assessed by ex vivo production ofIFN-gamma; and/or

5. Increased resistance to proteolysis.

Some embodiments relate to peptide sequences, including analogs, whereinthe amino acids of the sequence are referred to with a positiondesignator, for example P1, P2, P3, PΩ, etc. In addition, the peptidesequences can be referred to as including a P0 and/or PΩ+1 designator.In some aspects, P0 can be X, XX, or XXX, wherein X is any amino acid orno amino acid. Similarly, in some aspects, PΩ+1 can be X, XX, or XXX,wherein X is any amino acid or no amino acid. Thus, for example, XXX canmean any combination of any amino acid residues or no amino acidresidues. Thus, these embodiments can encompass polypeptides having upto three additional amino acids (with any combination of amino acidresidues) on the N-terminus or C-terminus of the specified sequence.Also, in some aspects, the embodiments can encompass no additional aminoacid residues on the N-terminus or the C-terminus.

The MHC residues responsible for forming binding pockets, and whichbinding pockets accommodate epitope anchor residues and thus define thebinding specificity of the MHC molecule, are well understood in the art.One compilation of such information is found at the FIMM (FunctionalImmunology) web site at the hypertext transfer protocol (http://)“sdmc.lit.org.sg:8080/fimm/”, which is hereby incorporated by referencein its entirety. (See also Schönbach C., Koh J. L. Y., Sheng X., WongL., and V. Brusic. FIMM, a database of functional molecular immunology.Nucleic Acids Research, 2000, Vol. 28, No. 1 222-224; and Schönbach C.,Koh J L, Flower D R, Wong L., and Brusic V. FIMM, a database offunctional molecular immunology; update 2002. Nucleic Acids Research,2002, Vol. 30, No. 1 226-229; each of which is hereby incorporated byreference in its entirety).

The phrase “liberation sequence,” as used herein, refers to a peptidecomprising or encoding an epitope or an analog, which is embedded in alarger sequence that provides a context allowing the epitope or analogto be liberated by immunoproteasomal processing, directly or incombination with N-terminal trimming or other physiologic processes. Insome aspects, the analog or epitope can be designed or engineered.

Other embodiments relate to epitope arrays and other polypeptides thatinclude the epitope analog sequences that can be processed to liberatethe analog. Further embodiments relate to nucleic acids, particularlyDNA plasmids, encoding such polypeptides, or simply an analog, and theirexpression therefrom. The analogs, the polypeptides comprising them, andthe encoding nucleic acids can all be components of immunogeniccompositions, particularly compositions suitable for intralymphaticdelivery, all of which relate to further embodiments.

Peptide analogs with improved immunologic properties can be designed bymodifying the anchor residues involved in the interaction with MHCmolecules, so as to increase the binding and stabilize the formation ofMHC-peptide complexes. Such modifications can be guided by knowledge ofthe binding motif or preferred anchor residues of the restricting MHCmolecule. There further exist various rules, indexes and algorithms thatcan be used to predict the properties of analogs bearing varioussubstitutions with the limitation that the substitution is selected fromthe standard set of genetically encodable amino acids.

However, there are no databases or algorithms to predict the outcome ofreplacing anchor residues with non-standard amino acids and theirusefulness is previously not well explored. It is herein disclosed thatthe non-standard amino acids norleucine (Nle) and norvaline (Nva) can beadvantageously substituted into the anchor residue positions ofMHC-binding peptides. It is preferred that they be placed in a positionfavorably occupied by a hydrophobic or a large amino acid, especially I,L, or V.

MHC-binding motifs are generally defined in terms of preferred residueside chains at nominal positions within a span of 8 to 10 amino acids(see for example Rammensee et al., “MHC Ligands and Peptide Motifs,”(Molecular Biology Intelligence Unit), Springer-Verlag, Germany, 1997Landes Bioscience, Austin, Tex.; and Parker, et al., “Scheme for rankingpotential HLA-A2 binding peptides based on independent binding ofindividual peptide side-chains,” J. Immunol. 152:163-175. Websitealgorithms are also available which can be used to predict MHC binding.See for example, the world wide web page of Hans-Georg Rammensee, JuttaBachmann, Niels Emmerich, Stefan Stevanovic: SYFPEITHI: An InternetDatabase for MHC Ligands and Peptide Motifs (hypertext transfer protocolaccess via: syfpeithi.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm)and “bimas.dcrt.nih.gov/molbio/hla_bind.” For class I-restrictedepitopes the C-terminal position, PΩ, is typically a primary anchor. The2nd position, P2, is also often a primary anchor or, alternatively, P3and/or P5 can serve this role. Positions P2 through P7 have all beenrecognized as secondary or auxiliary anchor positions for one or anotherMHC (see Rammensee et al., and see Table 6 from U.S. Patent ApplicationPublication No. 20030215425 (U.S. patent application Ser. No.10/026,066, filed on Dec. 7, 2001, entitled EPITOPE SYNCHRONIZATION INANTIGEN PRESENTING CELLS; which is incorporated herein by reference inits entirety for all of its disclosure). For class II-restrictedepitopes, P1, P4, P6, P7, and P9 have been recognized as anchorpositions. The foregoing is intended as a general guide and should beconsidered exemplary and not exhaustive or limiting. Many analyses andcompilations of binding motifs, anchor residues, and the like areavailable in the scientific and patent literature and over the internet.Their conventions and results further provide those of skill in the artuseful guidance regarding the design of epitope analogs, when coupledwith the teaching herein.

The length of the peptide actually bound to the presenting MHC moleculecan be longer than the nominal motif sequence. The ends of the bindingcleft for class II MHC molecules are open so that the bound peptide canbe extended at either end of the core motif. In contrast, the bindingcleft is closed at both ends in class I MHC molecules so that the endsof the bound peptide must generally correspond to the motif, however,significant variation in length can be accommodated through bulging andfolding of the central region of the bound peptide, so that peptides ofup to at least about 14 amino acids in length can be presented (see forexample Probst-Kepper, M. et al., J. Immunol. 173:5610-5616, 2004).

Epitope analogs can have improved K_(on) and K_(off) related to theinteraction with class I MHC molecules, as well as preserved orincreased interaction with T cell receptors recognizing the originalepitope, modified or improved in vivo or ex vivo activity reflected inenhanced expansion of specific T cell populations, improved cytokineproduction by specific T cells, or in vivo or in vitro cytotoxicityagainst targets carrying natural epitopes, mediated by T cells thatreacted with the peptide. In addition, such analogs can interact in amore optimal fashion with multiple distinct MHC class I molecules.

Such peptide analogs with improved immune properties can encompass oneor multiple substitutions, including one or multiple non-standard aminoacid substitutions. Among non-standard amino acid substitutions,substitutions for primary anchor residues consisting of norvaline ornorleucine are preferred because, as exemplified below, they can notonly improve the interaction with MHC class I, but can also preservecross-reactivity with TCR specific for the native epitope and showimproved in vivo immune profile. For example, mutating the P2 amino acidresidue from A, L or V to norvaline or norleucine improved immuneproperties and is thus preferred. In addition, modifying the C terminalresidue to norvaline or preferably norleucine, improved immune featuresof the analogs. In addition, analogs that encompass multiplesubstitutions at primary and/or secondary anchor residues includingnorvaline and/or norleucine at P2 or PΩ can be associated with improvedimmune properties.

Certain uses of norvaline (Nva) and norleucine (Nle) are mentioned inU.S. Pat. No. 6,685,947; PCT Publication Nos. WO 03/076585 A2 and WO01/62776 A1; and U.S. Patent Publication No. 20040253218A1. None ofthese references teaches the general usefulness of Nva or Nlesubstituted at an anchor position of a MHC-biding peptide to improve animmunological property. The '218 publication teaches that thesubstituted residues should be incorporated at TCR-interacting positionsand not at MHC-interacting positions.

In still another embodiment of the invention, the peptide is an analogof a peptide derived from an NS-specific antigen that is immunogenic butnot encephalitogenic. The most suitable peptides for this purpose arethose in which an encephalitogenic self-peptide is modified at theT-cell receptor (TCR) binding site and not at the MHC binding site(s),so that the immune response is activated but not anergized (Karin etal., 1998; Vergelli et al, 1996).

Others have also suggested a variety of non-standard amino acids may besubstituted without destroying the usefulness of the peptide (see forexample WO 02/102299A). Non-standard amino acids have also been used toinvestigate interaction between TCR and the MHC-peptide complex (see forexample WO 03/076585A and Sasada, T. et al. Eur. J. Immunol.30:1281-1289, 2000). Baratin showed some usefulness of Nle and Abu atnon-anchor positions and Nle at an anchor position in a p53 peptidepresented by the murine MHC molecule H2-Db (J. Peptide Sci. 8:327-334,2002). Each of these forgoing documents is hereby incorporated byreference in its entirety.

HLA-A2.1-restricted peptides incorporating Nle disclosed in WO 01/62776are derived from CEA, p53, and MAGE-3. In the CEA peptide I(Nle)GVLVGVand the p53 peptide S(Nle)PPPGTRV (SEQ ID NO. 101), Nle is present atthe P2 position. No teaching about the general usefulness of norleucineis provided and no disclosure is provided indicating how or if thesesubstitutions altered the properties of the analogs as compared to thenative sequence.

Some of the instant embodiments relate to epitope analogs thatincorporate Nva and/or Nle at a position promoting binding to MHC. Someembodiments specifically exclude the use Nle and/or Nva inHLA-A2.1-restricted epitopes, HLA-A2.1 epitopes from CEA, p53, and/orMAGE-3, or other peptides derived from MAGE-3, CEA, and/or p53. In someembodiments, one or more of the specific sequences as disclosed in theabove patent references are specifically excluded. In other embodiments,analogs of murine or other non-human MHC-restricted epitopes areexcluded. Other exemplary embodiments include the use of Nle and/or Nvaat P3, P5, and/or PΩ anchor positions, in an auxiliary anchor position,to make an analog of a non-A2- or non-A2.1-HLA restricted epitope, in ananchor position of a peptide that is not derived from an oncogene oroncofetal protein, and in an anchor position of a peptide derived from aCT antigen.

In general, such analogs may be useful for immunotherapy and/orprophylaxis of various diseases, such as infectious, cancerous orinflammatory, as single agents or in combination therapies. This is dueto their optimized interaction with MHC molecules and T cell receptorskey to onset and regulation of immune responses.

Analog Production

The analogs may be produced using any method known to one of skill inthe art, including manufacturing the peptides using a method of peptidesynthesis or expressing nucleic acids that code for the desired peptideanalogs. Thus, when the analogs include one or more non-standard aminoacids, it is more likely that they will be produced by a method ofpeptide production. When the analogs include only one or moresubstitutions with standard amino acids, they can be expressed from anexpression vector using any method known to one of skill in the art.Alternatively, the peptides can be expressed using a method of genetherapy.

Analog Testing

To evaluate usefulness and/or activity, and/or improved properties ofthe analogs and to compare the analogs in any way to the wild-type, oneor more of the following assays were conducted: peptide binding affinityfor HLA-A*0201; peptide-HLA-A*0201 complex stability assay; across-reactivity assay (recognition of peptide analogs by wild-type,peptide-specific CTL or recognition of wild-type peptide by CTLgenerated using peptide analogs); an immunogenicity assay, such as anIFN-γ secretion assay, a cytotoxicity assay, and/or an Elispot assay; anantigenicity assay, such as an in vitro tumor cell lysis assay, an exvivo tumor cell lysis, and an in vivo tumor cell lysis; and aproteolysis assays to identify increased resistance to proteolysis.Details of exemplary assays are presented in the Examples below.

The Using the above methodologies, useful and/or improved analogs wereidentified. To be useful, an analog may not necessarily be found to beimproved in the identified assays. For example, a useful peptide cancontain other properties such as being useful in a tolerized patient orresistant to proteolysis. To be improved, a peptide can be found to havea clear improvement in binding to the TCR, binding to the MHC molecule,and an improved immune response or any other biological activity. Insome instances, a useful peptide does not appear to be improved whenusing a murine test system, but because of the differences in the humanimmune system, is determined to be improved when tested in a human. Insome cases, the usefulness can stem from a potential to break tolerancein a tolerized human. In some instances, the usefulness can stem fromthe ability to use the peptide as a base for further substitutions toidentify improved analogs.

Uses of the Analogs

Useful methods for using the disclosed analogs in inducing, entraining,maintaining, modulating and amplifying class I MHC-restricted T cellresponses, and particularly effector and memory CTL responses toantigen, are described in U.S. Pat. No. 6,994,851 (Feb. 7, 2006) andU.S. Pat. No. 6,977,074 (Dec. 20, 2005) both entitled “A Method ofInducing a CTL Response”; U.S. Provisional Application No. 60/479,393,filed on Jun. 17, 2003, entitled “METHODS TO CONTROL MHC CLASSI-RESTRICTED IMMUNE RESPONSE”; and U.S. patent application Ser. No.10/871,707 (Pub. No. 2005 0079152) and Provisional U.S. PatentApplication No. 60/640,402 filed on Dec. 29, 2004, both entitled“METHODS TO ELICIT, ENHANCE AND SUSTAIN IMMUNE RESPONSES AGAINST MHCCLASS I-RESTRICTED EPITOPES, FOR PROPHYLACTIC OR THERAPEUTIC PURPOSE”.The analogs can also be used in research to obtain further optimizedanalogs. Numerous housekeeping epitopes are provided in U.S. applicationSer. No. 10/117,937, filed on Apr. 4, 2002 (Pub. No. 20030220239 A1),and U.S. application Ser. No. 10/657,022 (20040180354), and in PCTApplication No. PCT/US2003/027706 (Pub. No. WO04022709A2), filed on Sep.5, 2003; and U.S. Provisional Application Nos. 60/282,211, filed on Apr.6, 2001; 60/337,017, filed on Nov. 7, 2001; 60/363,210 filed on Mar. 7,2002; and 60/409,123, filed on Sep. 5, 2002; each of which applicationsis entitled “EPITOPE SEQUENCES”. The analogs can further be used in anyof the various modes described in those applications. Epitope clusters,which can comprise or include the instant analogs, are disclosed andmore fully defined in U.S. patent application Ser. No. 09/561,571, filedon Apr. 28, 2000, entitled EPITOPE CLUSTERS. Methodology for using anddelivering the instant analogs is described in U.S. patent applicationSer. Nos. 09/380,534 and 6977074 (Issued Dec. 20, 2005) and in PCTApplication No. PCTUS98/14289 (Pub. No. WO9902183A2), each entitled A“METHOD OF INDUCING A CTL RESPONSE”. Beneficial epitope selectionprinciples for such immunotherapeutics are disclosed in U.S. patentapplication Ser. No. 09/560,465, filed on Apr. 28, 2000, Ser. No.10/026,066 (Pub. No. 20030215425 A1), filed on Dec. 7, 2001, and Ser.No. 10/005,905 filed on Nov. 7, 2001, all entitled “EPITOPESYNCHRONIZATION IN ANTIGEN PRESENTING CELLS”; U.S. Pat. No. 6,861,234(issued 01-Mar.-2005; application Ser. No. 09/561,074), entitled “METHODOF EPITOPE DISCOVERY”; Ser. No. 09/561,571, filed Apr. 28, 2000,entitled EPITOPE CLUSTERS; Ser. No. 10/094,699 (Pub. No. 20030046714A1), filed Mar. 7, 2002, entitled “ANTI-NEOVASCULATURE PREPARATIONS FORCANCER”; application Ser. No. 10/117,937 (Pub. No. 20030220239 A1) andPCTUS02/11101 (Pub. No. WO02081646A2), both filed on Apr. 4, 2002, andboth entitled “EPITOPE SEQUENCES”; and application Ser. No. 10/657,022and PCT Application No. PCT/US2003/027706 (Pub. No. WO04022709A2), bothfiled on Sep. 5, 2003, and both entitled “EPITOPE SEQUENCES”. Aspects ofthe overall design of vaccine plasmids are disclosed in U.S. patentapplication Ser. No. 09/561,572, filed on Apr. 28, 2000, entitled“EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS” andSer. No. 10/292,413 (Pub. No. 20030228634 A1), filed on Nov. 7, 2002,entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATEDANTIGENS AND METHODS FOR THEIR DESIGN”; Ser. No. 10/225,568 (Pub No.2003-0138808), filed on Aug. 20, 2002, PCT Application No.PCT/US2003/026231 (Pub. No. WO 2004/018666), filed on Aug. 19, 2003,both entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATEDANTIGENS”; and U.S. Pat. No. 6,709,844, entitled “AVOIDANCE OFUNDESIRABLE REPLICATION INTERMEDIATES IN PLASMID PROPAGATION”. Specificantigenic combinations of particular benefit in directing an immuneresponse against particular cancers are disclosed in Provisional U.S.patent Application No. 60/479,554, filed on Jun. 17, 2003 and U.S.patent application Ser. No. 10/871,708, filed on Jun. 17, 2004 and PCTPatent Application No. PCT/US2004/019571 (Pub. No. WO 2004/112825), allentitled “COMBINATIONS OF TUMOR-ASSOClATED ANTIGENS IN VACCINES FORVARIOUS TYPES OF CANCERS”. Antigens associated with tumor neovasculature(e.g., PSMA, VEGFR2, Tie-2) are also useful in connection with cancerousdiseases, as is disclosed in U.S. patent application Ser. No. 10/094,699(Pub. No. 20030046714 A1), filed Mar. 7, 2002, entitled“ANTI-NEOVASCULATURE PREPARATIONS FOR CANCER”. Methods to trigger,maintain, and manipulate immune responses by targeted administration ofbiological response modifiers are disclosed U.S. Provisional ApplicationNo. 60/640,727, filed on Dec. 29, 2004. Methods to bypass CD4+ cells inthe induction of an immune response are disclosed in U.S. ProvisionalApplication No. 60/640,821, filed on Dec. 29, 2004. Exemplary diseases,organisms and antigens and epitopes associated with target organisms,cells and diseases are described in U.S. Pat. No. 6,977,074 (issued Dec.20, 2005) filed Feb. 2, 2001 and entitled “METHOD OF INDUCING A CTLRESPONSE”. Exemplary methodology is found in U.S. ProvisionalApplication No. 60/580,969, filed on Jun. 17, 2004, and U.S. PatentApplication No. 2006-0008468-A1, published on Jan. 12, 2006, bothentitled “COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN DIAGNOSTICS FORVARIOUS TYPES OF CANCERS”. Methodology and compositions are alsodisclosed in U.S. Provisional Application No. 60/640,598, filed on Dec.29, 2004, entitled “COMBINATIONS OF TUMOR-ASSOClATED ANTIGENS INCOMPOSITIONS FOR VARIOUS TYPES OF CANCER”. The integration of diagnostictechniques to assess and monitor immune responsiveness with methods ofimmunization including utilizing the instant analogs is discussed morefully in Provisional U.S. Patent Application No. 60/580,964 filed onJun. 17, 2004 and U.S. Patent Application No. US-2005-0287068-A1,published on Dec. 29, 2005) both entitled “IMPROVED EFFICACY OF ACTIVEIMMUNOTHERAPY BY INTEGRATING DIAGNOSTIC WITH THERAPEUTIC METHODS”.Immunogenic polypeptide encoding vectors are disclosed in U.S. patentapplication Ser. No. 10/292,413 (Pub. No. 20030228634 A1), filed on Nov.7, 2002, entitled EXPRESSION VECTORS ENCODING EPITOPES OFTARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN, and in U.S.Provisional Application No. 60/691,579, filed on Jun. 17, 2005, and thecorresponding U.S. patent application Ser. No. ______(Attorney DocketNo. MANNK. 053A filed on the same date as the present application) bothentitled “METHODS AND COMPOSITIONS TO ELICIT MULTIVALENT IMMUNERESPONSES AGAINST DOMINANT AND SUBDOMINANT EPITOPES EXPRESSED ON CANCERCELLS AND TUMOR STROMA”). Additional useful disclosure, includingmethods and compositions of matter, is found in U.S. ProvisionalApplication No. 60/691,581, filed on Jun. 17, 2005, entitled“MULTIVALENT ENTRAIN-AND-AMPLIFY IMMUNOTHERAPEUTICS FOR CARCINOMA.”Further methodology, compositions, peptides, and peptide analogs aredisclosed in U.S. Provisional Application Nos. 60/581,001 and60/580,962, both filed on Jun. 17, 2004, and respectively entitled“SSX-2 PEPTIDE ANALOGS” and “NY-ESO PEPTIDE ANALOGS.” Each of theapplications and patents mentioned in the above paragraphs is herebyincorporated by reference in its entirety for all that it teaches.Additional analogs, peptides and methods are disclosed in U.S. PatentApplication Publication No 20060063913, entitled “SSX-2 PEPTIDEANALOGS”; and U.S. Patent Publication No. 2006-0057673 A1, published onMar. 16, 2006, entitled “EPITOPE ANALOGS”; and PCT ApplicationPublication No. WO/2006/009920, entitled “EPITOPE ANALOGS”; all filed onJun. 17, 2005, as well as in U.S. Provisional Patent Application No.60/691,889, filed on Jun. 17, 2005 entitled EPITOPE ANALOGS; and U.S.patent application No. ______, entitled PRAME PEPTIDE ANALOGUES (AttyDocket No. MANNK.052A), U.S. patent application No. ______, entitledPSMA PEPTIDE ANALOGUES (Atty Docket No. MANNK.052A2), and U.S. patentapplication No. ______, entitled MELANOMA ANTIGEN PEPTIDE ANALOGUES(Atty Docket No. MANNK.052A3), each of which is hereby incorporated byreference in its entirety. Exemplary immunogenic products are disclosedin U.S. Provisional Patent Application No. 60/691,581, filed on Jun. 17,2005 and U.S. patent application No. ______(Atty. Docket No.MANNK.054A), filed on date even with the instant application, eachentitled MULTIVALENT ENTRAIN-AND-AMPLIFY IMMUNOTHERAPEUTICS FORCARCINOMA, and each incorporated by reference in its entirety. Furthermethodology and compositions are disclosed in U.S. ProvisionalApplication No. 60/581,001, filed on Jun. 17, 2004, entitled “SSX-2PEPTIDE ANALOGS”, and to U.S. Provisional Application No. 60/580,962,filed on Jun. 17, 2004, entitled “NY-ESO PEPTIDE ANALOGS”; each of whichis incorporated herein by reference in its entirety. Other applicationsthat are expressly incorporated herein by reference are: U.S. patentapplication Ser. No. 11/156,253 (Publication No.), filed on Jun. 17,2005, entitled “SSX-2 PEPTIDE ANALOGS”; U.S. patent application Ser. No.11/155,929, filed on Jun. 17, 2005, entitled “NY-ESO-1 PEPTIDE ANALOGS”(Publication No.); U.S. patent application Ser. No. 11/321,967, filed onDec. 29, 2005, entitled “METHODS TO TRIGGER, MAINTAIN AND MANIPULATEIMMUNE RESPONSES BY TARGETED ADMINISTRATION OF BIOLOGICAL RESPONSEMODIFIERS INTO LYMPHOID ORGANS”; U.S. patent application Ser. No.11/323,572, filed on Dec. 29, 2005, entitled “METHODS TO ELICIT ENHANCEAND SUSTAIN IMMUNE REPONSES AGAINST MCH CLASS I RESTRICTED EPITOPES, FORPROPHYLACTIC OR THERAPEUTIC PURPOSES”; U.S. patent application Ser. No.11/323,520, filed Dec. 29, 2005, entitled “METHODS TO BYPASS CD4+ CELLSIN THE INDUCTION OF AN IMMUNE RESPONSE”; U.S. patent application Ser.No. 11/323,049, filed Dec. 29, 2005, entitled “COMBINATION OFTUMOR-ASSOClATED ANTIGENS IN COMPOSITIONS FOR VARIOUS TYPES OF CANCERS”;U.S. patent application Ser. No. 11,323,964, filed Dec. 29, 2005,entitled “COMBINATIONS OF TUMOR-ASSOClATED ANTIGENS IN DIAGNOSTICS FORVARIOUS TYPES OF CANCERS.” As an example, without being limited theretoeach reference is incorporated by reference for what it teaches aboutclass I MHC-restricted epitopes, analogs, the design of analogs, uses ofepitopes and analogs, methods of using and making epitopes, the designand use of nucleic acid vectors for their expression, and formulations.

Antigens

There are many antigens, epitopes of which can be recognized by T cellsin an MHC-restricted manner, for which manipulation of an immuneresponse directed against them has therapeutic or prophylacticpotential. The principles for making analogs of MHC-binding peptidesdescribed herein are generally applicable to any of these antigens andtheir epitopes. A particular focus of the present disclosure is epitopesfrom the tumor-associated antigens (TuAA) SSX-2, NY-ESO-1, PRAME, PSMA,tyrosinase, and Melan-A.

SSX-2, also know as Hom-Mel-40, is a member of a family of highlyconserved cancer-testis antigens (Gure, A. O. et al. Int. J. Cancer72:965-971, 1997, which is hereby incorporated by reference in itsentirety). Its identification as a TuAA antigen is taught in U.S. Pat.No. 6,025,191 entitled “ISOLATED NUCLEIC ACID MOLECULES THAT ENCODE AMELANOMA SPECIFIC ANTIGEN AND USES THEREOF”, which is herebyincorporated by reference in its entirety. Cancer-testis antigens arefound in a variety of tumors, but are generally absent from normal adulttissues except testis. SSX-2 is expressed in many different types oftumors, including synovial sarcomas, melanoma, head and neck cancers,breast, colon and ovarian cancers. In addition to its widespreadexpression in a variety of cancers, it is also immunogenic in patientswith late stage disease. Further, there is evidence of spontaneoushumoral and cellular immune responses towards this antigen in metastatictumor patients (Ayyoub M, et al., Cancer Res. 63(17): 5601-6, 2003;Ayyoub M, et al. J Immunol. 168(4): 1717-22, 2002), which isincorporated herein by reference in its entirety. Two HLA-A2 restrictedT cell epitopes have been identified recently using reverse T-cellimmunology, namely SSX-2₄₁₋₄₉ (Ayyoub M, et al. J. Immunol. 168(4):1717-22, 2002; U.S. Pat. No. 6,548,064, entitled “ISOLATED PEPTIDESCONSISTING OF AMINO ACID SEQUENCES FOUND IN SSX OR NY-ESO-1 MOLECULES,THAT BIND TO HLA MOLECULE”; U.S. patent application Ser. No. 10/117,937(Publication No. US 2003-0220239 A1), entitled “EPITOPE SEQUENCES”) andSSX-2₁₀₃₋₁₁₁ (Wagner C, et al. Cancer Immunity 3:18, 2003), each ofwhich is incorporated herein by reference in its entirety. The C-terminiof both epitopes can be efficiently generated by in vitro proteasomedigestion. Isolated HLA-A*0201/SSX-2₄₁₋₄₉ multimer⁺ CD8⁺ T cells fromtumor-infiltrated lymph nodes of SSX-2 positive patients exhibited highfunctional avidity and can effectively recognize SSX-2 positive tumors;however, the spontaneously occurring immunological responses were notsufficient for stopping tumor growth, possibly because these immuneresponse did not develop until fairly late in the disease progression,and the activated T cells were not numerous enough. U.S. Pat. No.6,548,064 (which is incorporated herein by reference in its entirety)further describes substituting a T or A residue at both the P2 and PΩposition of an SSX-2 epitope.

NY-ESO-1 is a cancer-testis antigen found in a wide variety of tumorsand is also known as CTAG-1 (Cancer-Testis Antigen-1) and CAG-3 (CancerAntigen-3). NY-ESO-1 as a tumor-associated antigen (TuAA) is disclosedin U.S. Pat. No. 5,804,381 entitled “ISOLATED NUCLEIC ACID MOLECULEENCODING AN ESOPHAGEAL CANCER ASSOCIATED ANTIGEN, THE ANTIGEN ITSELF,AND USES THEREOF,” which is hereby incorporated by reference in itsentirety. Paralogous locus encoding antigens with extensive sequenceidentity, LAGE-1a/s and LAGE-1b/L, have been disclosed in publiclyavailable assemblies of the human genome, and have been concluded toarise through alternate splicing. Additionally, CT-2 (or CTAG-2,Cancer-Testis Antigen-2) appears to be either an allele, a mutant, or asequencing discrepancy of LAGE-1b/L. Due to the extensive sequenceidentity, many epitopes from NY-ESO-1 can also induce immunity to tumorsexpressing these other antigens. The proteins are virtually identicalthrough amino acid 70. From residues 71-134 the longest run of identitybetween NY-ESO-1 and LAGE is 6 residues, but potentially cross-reactivesequences are present. From residues 135-180, NY-ESO and LAGE-1a/s areidentical except for a single residue, but LAGE-1b/L is unrelated due tothe alternate splice. The CAMEL and LAGE-2 antigens appear to derivefrom the LAGE-1 mRNA, but from alternate reading frames, thus givingrise to unrelated protein sequences. More recently, GenBank AccessionAF277315.5, Homo sapiens chromosome X clone RP5-865E18, RP5-1087L19,complete sequence, which is incorporated herein by reference in itsentirety, reports three independent loci in this region that are labeledas LAGE1 (corresponding to CTAG-2 in the genome assemblies), plusLAGE2-A and LAGE2-B (both corresponding to CTAG-1 in the genomeassemblies).

NY-ESO-1₅₇₋₁₆₅ is identified as an HLA-A2 restricted epitope in U.S.Pat. No. 6,274,145 entitled “ISOLATED NUCLEIC ACID MOLECULE ENCODINGCANCER ASSOClATED ANTIGEN, THE ANTIGEN ITSELF, AND USES THEREOF”, andU.S. patent application Ser. No. 10/117,937 (Pub. No. 20030220239)entitled “EPITOPE SEQUENCES” reports that this C-terminus is generatedby the housekeeping proteasome in an in vitro assay. Analogssubstituting A, V, L, I, P, F, M, W, or G at PΩ, alone or in combinationwith A at another position, are disclosed in U.S. Pat. Nos. 6,417,165and 6,605,711, both entitled “NY-ESO-1-PEPTIDE DERIVATIVES AND USESTHEREOF”. Each of the references described in this paragraph isincorporated herein by reference in its entirety.

PRAME, also known as MAPE, DAGE, and OIP4, was originally observed as amelanoma antigen. Subsequently, it has been recognized as a CT antigen,but unlike many CT antigens (e.g., MAGE, GAGE, and BAGE) it is expressedin acute myeloid leukemias. PRAME is a member of the MAPE family whichconsists largely of hypothetical proteins with which it shares limitedsequence similarity. The usefulness of PRAME as a TuAA is taught in U.S.Pat. No. 5,830,753 entitled “ISOLATED NUCLEIC ACID MOLECULES CODING FORTUMOR REJECTION ANTIGEN PRECURSOR DAGE AND USES THEREOF”, which ishereby incorporated by reference in its entirety. U.S. patentapplication Ser. No. 10/181,499 (Publication No. US 2003-0186355 A1),entitled “METHODS FOR SELECTING AND PRODUCING T CELL PEPTIDE EPITOPESAND VACCINES INCORPORATING SAID SELECTED EPITOPES” (which isincorporated herein by reference in its entirety) identifies a varietyof potential epitopes, including PRAME₄₂₅₋₄₃₃, using in vitro digestionwith immunoproteasome.

PSMA (prostate-specific membranes antigen), a TuAA described in U.S.Pat. No. 5,538,866 entitled “PROSTATE-SPECIFIC MEMBRANES ANTIGEN,” whichis hereby incorporated by reference in its entirety, is expressed bynormal prostate epithelium and, at a higher level, in prostatic cancer.It has also been found in the neovasculature of non-prostatic tumors.PSMA can thus form the basis for vaccines directed to both prostatecancer and to the neovasculature of other tumors. This later concept ismore fully described in U.S. Patent Publication No. 20030046714; PCTPublication No. WO 02/069907; and a provisional U.S. Patent applicationNo. 60/274,063 entitled “ANTI-NEOVASCULAR VACCINES FOR CANCER”, filedMar. 7, 2001, and U.S. application Ser. No. 10/094,699 (Publication No.US 2003-0046714 A1), filed on Mar. 7, 2002, entitled “ANTI-NEOVASCULARPREPARATIONS FOR CANCER,” each of which are hereby incorporated byreference in its entirety. The teachings and embodiments disclosed insaid publications and applications provide supporting principals andembodiments related to and useful in connection with the presentinvention. Briefly, as tumors grow they recruit ingrowth of new bloodvessels. This is understood to be necessary to sustain growth as thecenters of unvascularized tumors are generally necrotic and angiogenesisinhibitors have been reported to cause tumor regression. Such new bloodvessels, or neovasculature, express antigens not found in establishedvessels, and thus can be specifically targeted. By inducing CTL againstneovascular antigens the vessels can be disrupted, interrupting the flowof nutrients to (and removal of wastes from) tumors, leading toregression.

Alternate splicing of the PSMA mRNA also leads to a protein with anapparent start at Met₅₈, thereby deleting the putative membrane anchorregion of PSMA as described in U.S. Pat. No. 5,935,818, entitled“ISOLATED NUCLEIC ACID MOLECULE ENCODING ALTERNATIVELY SPLICEDPROSTATE-SPECIFIC MEMBRANES ANTIGEN AND USES THEREOF” which is herebyincorporated by reference in its entirety. A protein termed PSMA-likeprotein, Genbank accession number AF261715, which is hereby incorporatedby reference in its entirety, is nearly identical to amino acids 309-750of PSMA and has a different expression profile. Thus, the more preferredepitopes are those with an N-terminus located from amino acid 58 to 308.PSMA₂₈₈₋₂₉₇ was identified as possessing an HLA-A2 binding motif in WO01/62776, entitled “HLA BINDING PEPTIDES AND THEIR USES”, which ishereby incorporated by reference in its entirety. Its production invitro by digestion with a housekeeping proteasome and actual binding toHLA-A2 was disclosed in U.S. Patent Application Publication No.20030220239 entitled “EPITOPE SEQUENCES”.

Tyrosinase is a melanin biosynthetic enzyme that is considered one ofthe most specific markers of melanocytic differentiation. Tyrosinase isexpressed in few cell types, primarily in melanocytes, and high levelsare often found in melanomas. The usefulness of tyrosinase as a TuAA istaught in U.S. Pat. No. 5,747,271, entitled “METHOD FOR IDENTIFYINGINDIVIDUALS SUFFERING FROM A CELLULAR ABNORMALITY SOME OF WHOSE ABNORMALCELLS PRESENT COMPLEXES OF HLA-A2/TYROSINASE DERIVED PEPTIDES, ANDMETHODS FOR TREATING SAID INDIVIDUALS,” which is hereby incorporated byreference in its entirety.

Melan-A, also called MART-1 (Melanoma Antigen Recognized by T cells), isanother melanin biosynthetic protein expressed at high levels inmelanomas. The usefulness of Melan-A/MART-1 as a TuAA is taught in U.S.Pat. Nos. 5,874,560 and 5,994,523, both entitled “MELANOMA ANTIGENS ANDTHEIR USE IN DIAGNOSTIC AND THERAPEUTIC METHODS”, as well as U.S. Pat.No. 5,620,886, entitled “ISOLATED NUCLEIC ACID SEQUENCE CODING FOR ATUMOR REJECTION ANTIGEN PRECURSOR PROCESSED TO AT LEAST ONE TUMORREJECTION ANTIGEN PRESENTED BY HLA-A2”, each of which is herebyincorporated by reference in its entirety. The immunodominant HLA-A2restricted epitope from this TuAA is Melan-A₂₆₋₃₅. It has been shown tobe produced by the housekeeping proteasome (Morel, S. et al., Immunity12:107-117, 2000, which is hereby incorporated by reference in itsentirety). Various analogs incorporating standard amino acids, includingan improved analog substituting L at P2, are disclosed in U.S. Pat. No.6,025,470, entitled “ISOLATED NONA-AND DECAPEPTIDES WHICH BIND TO HLAMOLECULES, AND THE USE THEREOF”, which is hereby incorporated byreference in its entirety. The use of analogs incorporating non-standardamino acids with a primary goal of improving biochemical stability isreported by Blanchet, J.-S. et al., J. Immunol. 167:5852-5861, 2001,which is hereby incorporated by reference in its entirety.

SSX-2 41-49 Analogs

As noted above, the natural immune response to SSX-2 in cancer patients,including the response to SSX-2₄₁₋₄₉, may not be effective incontrolling cancer. Additionally, wild-type SSX-2₄₁₋₄₉ is only amoderately immunogenic peptide, which can further limit its clinicalpotential. Stronger SSX-2 specific immune responses induced by the useof superagonist analogs results in clinical benefits for patients withSSX-2 positive tumors.

Thus, in one embodiment, the analogs can be used in compositions tostimulate the immune response of a subject to mount an immune responseagainst a target cell displaying the target antigen. The embodiment canhave utility in the treatment and prevention of neoplastic and viraldisease.

Because the wild-type SSX-2₄₁₋₄₉ is only a moderately immunogenicpeptide, which may prevent it from eliminating tumors effectively invivo, a method was used to de novo design SSX-2₄₁₋₄₉ variants that weremore potent or had a variety of improved properties. By using a moreimmunogenic SSX-2 analog peptide, it was possible to stimulate astronger immune response and/or to amplify the naturally occurringimmune response to achieve a better chance of clinical response. Thus,the binding properties (affinity and HLA-A*0201/peptide complexesstability), immunogenicity, antigenicity and cross-reactivity to thewild-type epitope were analyzed for each of the analogs to identify animproved property. In some embodiments, by improved property it is meantgenerally, that the analog can be better used for some purpose than thewild-type. Thus, the analog need not exhibit improved binding,stability, or activity to be improved and may even show a reducedability to mediate certain parts of the process, but still be improvedfor use in another way. For example, analogs that retain some activity,but not all activity can be better in human systems that are tolerizedto the wild-type antigen.

Previously, modifications of natural tumor-associated peptide epitopesby incorporating favorable anchor residues have generated analogs withimproved binding profiles with HLA molecules and enhancedimmunogenicity. One of the most successful examples is the A27L peptideanalog of Melan-A 26-35 epitope. Valmori et al., “Enhanced generation ofspecific tumor-reactive CTL in vitro by selected Melan-A/MART-1immunodominant peptide analogs,” J Immunol. 1998, 160(4): 1750-8, whichis hereby incorporated by reference in its entirety. The originalepitope failed to form a stable complex with HLA-A2 molecules because itlacked an optimum anchor amino acid residue at position 2. The modifiedA27L Melan A 26-35 peptide analog has demonstrated unequivocallyincreased binding profiles with HLA-A2 molecules and greaterimmunogenicity than its wild-type counterpart. Immunizing patients withthis analog generated strong T cell immune responses that were able torecognize the wild-type epitope presented at the cell surfaces. Similarmodifications have been obtained successfully with many othertumor-associated epitopes, such as GP100 209-217 (Parkhurst et al.,“Improved induction of melanoma-reactive CTL with peptides from themelanoma antigen gp100 modified at HLA-A*0201-binding residues,” JImmunol. 1996, 157(6): 2539-48; which is hereby incorporated byreference in its entirety), and Her-2 369-377 (Vertuani et al.,“Improved immunogenicity of an immunodominant epitope of the HER-2/neuprotooncogene by alterations of MHC contact residues,” J. Immunol. 2004,172(6): 3501-8; which is hereby incorporated by reference in itsentirety).

Methods are disclosed herein that can be used for the identification andproduction of analogs to a Synovial sarcoma X breakpoint 2 (SSX-2)wild-type sequence. Using the methods disclosed herein, a panel of 95novel SSX-2₄₁₋₄₉ analogs based on the wild-type sequence from aminoacids 241-249 were identified with a variety of improved properties. Theimproved properties include, but are not limited to, binding to class IMHC and T cell receptor (TCR) molecules, and biological responses suchas IFN-γ secretion, cytotoxicity, and tumor cell lysis. Peptides withimproved potency that retained cross-reactivity with the wild-typeepitope were also identified. Among these analogs, some weredemonstrated to be the superagonist variants of the wild-type SSX-2₄₁₋₄₉peptide, some of which were shown to have much higher affinity withHLA-A*0201 molecule, and the peptide-HLA complex possessed extendedstability. These analogs induced enhanced CTL immune responses in HHDtransgenic mice immunized with them. The resulting CTLs couldeffectively lyse A2+ and SSX-2+tumor cell lines both in vivo and invitro, indicating that the CTLs generated using the analogs were able torecognize the wild-type SSX-2₄₁₋₄₉ epitope that naturally presented atthe cell surfaces. In comparison with the wild-type SSX-2₄₁₋₄₉ epitope,the analogs are better candidates for the development of cancervaccines.

Accordingly, embodiments of the invention disclosed herein includefamilies of one or more peptides of 9 or 10 amino acids in lengthrelated by sequence to amino acids 41-49 of the human cancer testis (CT)antigen SSX-2 (SSX-2₄₁₋₄₉). The individual peptide embodiments have oneto several defined amino acid substitutions in the wild-type sequence.The substituted amino acids are, variously, other members of thestandard set of amino acids commonly genetically encoded, derivativesthereof, their D-stereoisomers, or other non-standard L-amino acids.These analogs are useful for investigating the interaction of thewild-type epitope with class I MHC and TCR molecules and othercomponents of the immune response, and for designing additional analogswith further optimized immunologic properties. Some embodiments of theanalogs have at least similar immunologic properties to the wild-typeepitope in the HLA-transgenic mouse model in which they have beentested. Such peptides can be useful in humans, as SSX-2 is aself-antigen to which a degree of tolerance may be expected, and theamino acid differences of the analogs can help to stimulate populationsof T cells that have avoided negative selection but are cross-reactivewith the wild-type epitope. Various peptide embodiments can have one ormore improved immunologic properties in that they possess greateraffinity for MHC or greater stability of binding to MHC, elicit greatercytokine production or require lower peptide concentrations to elicitsimilar cytokine production from T cells that recognize the wild-typeepitope, are more immunogenic, can induce or amplify a cross-reactivecytolytic response to the wild-type epitope, and/or can break tolerance.

In one embodiment, the analogs can have at least one substitution at aresidue selected from the group consisting of, P1, P2, P4, P6, P8, P9and P10. In a further embodiment, the analogs can have at least twosubstitutions at residues selected from the group consisting of: P1, P2,P4, P6, P8, P9 and P10. In a further embodiment, the analogs can have atleast three substitutions at residues selected from the group consistingof: P1, P2, P4, P6, P8, P9 and P10. In a further embodiment, the analogscan have substitutions at positions P2 and P9. In a further embodiment,the peptides can have substitutions at residues P1, P2, and P9. In afurther embodiment, the peptide analogs can have substitutions atresidues P1, P2, and P4. In a further embodiment, the peptide analogscan have substitutions at residues P1, P2, and P6. In a furtherembodiment, the peptide analogs can have substitutions at residues P1,P2, and P8. In one embodiment, two substitutions can produce improvedproperties. In a further embodiment, one substitution can produceimproved properties. In a further embodiment, three substitutions canproduce improved properties. In a further embodiment, the one or moresubstitutions can produce improved properties but are still recognizedby a TCR that recognizes the wild-type sequence (still cross-reactivewith the wild-type sequence).

One embodiment relates to epitope arrays and other polypeptidescomprising the epitope analog sequences that can be processed toliberate the analog. Further embodiments relate to nucleic acids,particularly DNA plasmids, encoding such polypeptides, or simply ananalog, and their expression therefrom. The analogs, the polypeptidescomprising them, and the encoding nucleic acids can all be components ofimmunogenic compositions, particularly compositions suitable forintralymphatic delivery, that constitute further embodiments.

Analog Design

Embodiments relate to SSX-2₄₁₋₄₉ peptides containing substitutions ofthe sequence KASEKIFYV (SEQ ID NO. 1) (See FIG. 1). In a furtherembodiment, the analog can be generally an analog of the SSX-2₄₁₋₅₀decamer peptide with the sequence KASEKIFYVY (SEQ ID NO. 1). Theresidues or amino acids that make up the peptide are referred to hereinas P1-P9 or P1-P10 to designate the position within the peptide asnumbered from the N— to the C-terminus, P1 corresponding to theN-terminal Lysine and P9 corresponding to the C-terminal Valine in thenonamer. Alternatively, the residues may be referred to by the primaryactivity of the molecule that they are involved in. For example, residueP2 is described as the N-terminal primary anchor molecule, while P9 (orP10 in the decamer) is described as the primary C-terminal anchor.Residues P4, P6 and P8 are primarily involved in TCR interactions.Substitutions can use any amino acids, including standard andnon-standard amino acids, known to one of skill in the art. A number ofexemplary amino acids are disclosed herein, however, the substitutionsdisclosed herein are not meant to be a list that includes all imaginedsubstitutions, but are exemplary of the substitutions that are possible.One of skill in the art may find a number of other non-standard aminoacids in catalogs and references that may be purchased or chemicallyproduced for use with the analogs herein.

A number of possible analogs were produced by modification of peptideanchor residues to achieve better HLA binding profiles and higher immuneresponses, including at the N-terminal primary anchor (P2 position), atthe N-terminal secondary anchor (P1 position), at the N-terminal primaryand secondary anchor (P1 and P2 positions), and at the N-terminalprimary/secondary anchor (P1 and P2 positions) and C-terminal primaryanchor (P9 position). Further, peptides with modifications at the anchorresidues and TCR contact residues were produced to circumvent T celltolerance for self-antigens, these modifications included modificationsat the N-terminal primary/secondary anchor (P1 and P2 positions) andsecondary TCR recognition sites (P4, P6 and/or P8 positions),modifications at the N-terminal primary/secondary anchors (P1 and P2position), and modifications at the C-terminal primary anchor (P9) andat secondary TCR recognition sites (P4, P6 and/or P8 positions).Further, decamer analogs were produced.

The choice of which residues would best produce analogs with improvedproperties involved analysis of studies of MHC peptide interactions,studies of TCR peptide interactions and previous analogs that were knownin the art. Some residues are primarily involved in a specificinteraction and some are secondarily or even tertiarily involved. Thus,the knowledge of how the residues are involved in the binding to thesemolecules was involved in the analysis. Further, some of the wild-typeresidues are preferred, meaning that they work well for the intendedinteraction, while others are non-preferred, meaning that they workpoorly for the interaction. Thus, in one embodiment, the non-preferredresidues can be substituted. For example, the valine at the C-terminusis generally a preferred anchor residue because it produces a stronginteraction with the HLA molecule and, thus, it was less preferred tosubstitute this residue. However, modifications of wild-typetumor-associated peptide epitopes by incorporating favorable anchorresidues have generated analogs with improved binding profiles with HLAmolecules and enhanced immunogenicity. One of the most successfulexamples is the A27L peptide analog of Melan-A 26-35 epitope (Valmori D,et al. J. Immunol. 160(4): 1750-8, 1998; which is hereby incorporated byreference in its entirety). The original epitope failed to form a stablecomplex with HLA-A2 molecules as it lacked an optimal anchor residue atposition 2. In contrast, the modified Melan A₂₆₋₃₅ A27L peptide analogdemonstrated unequivocally increased binding profiles with HLA-A2molecules and greater immunogenicity than its wild-type counterpart.Immunizing patients with this analog generated strong T cell immuneresponses that were able to recognize the wild-type epitope presented atthe cell surfaces. Similar modifications were obtained successfully withmany other tumor-associated epitopes such as GP100 209-217 (Parkhurst MR, et al. J Immunol. 157(6): 2539-48, 1996; which is hereby incorporatedby reference in its entirety), Her-2 369-377 (Vertuani S, et al. J.Immunol. 172(6): 3501-8, 2004; which is hereby incorporated by referencein its entirety).

The choice of how many residues to substitute involves a desire tosubstitute better residues while still retaining enough of the qualitiesof the epitope that it will still be recognized by T cells thatrecognize the wild-type epitope. Thus, in one embodiment, one or twosubstitutions can be made to the wild-type peptide. In a furtherembodiment, more than two substitutions can be made to the wild-typepeptide, while still retaining cross-reactivity with the wild-typepeptide.

Generally, the part of the peptide that is involved in TCR recognitionis desirably substituted to produce improved immunogenicity while stillcross-reacting with the wild-type epitope. In one example, a peptidethat shows increased immunogenicity is preferred. Because the P2position or second amino acid at the N-terminal end is believed to beprimarily involved in the process of producing improved immunogenicity,primarily through improved binding properties, it is a preferredsubstitution site and a number of modifications were made in theexemplary analogs to identify desirable substitutions. Similarconsiderations apply to the carboxy-terminal position, PΩ, which alsocan be important for MHC binding.

Thus, in one embodiment, the analog can include a substitution at the P2residue that substitutes a more hydrophobic residue for the wild-typealanine. In a further embodiment, the hydrophobic residue also canpossess a more bulky side chain. In a further embodiment, the residue atP1 can be substituted with a more hydrophobic residue. In a furtherembodiment, residues P1 and P2 both can be substituted with morehydrophobic residues. In further embodiments, at least one residue atP1, P2, and P9 can be substituted. In a further embodiment, at least tworesidues at P1, P2 and P9 can be substituted. In a further embodiment atleast two residues at P1, P2, P9, P4, and P6 can be substitutedincluding one or more residues involved in TCR binding.

In some embodiments, substitutions of those residues only secondarilyinvolved in binding to TCR or the MHC molecule can be advantageous. Forexample, substitution of secondary TCR binding amino acids can generateanalogs that still bind and produce a response and do not interfere withthe binding to the MHC molecule, but preferably overcome the toleranceissues of self-antigens. This is useful because a patient who has cancermay be partially tolerized to the antigen. Thus, in order to overcomethat tolerance, an analog that retains some activity can be preferableto an analog with more improved immunogenicity, because it will be lesslikely to be recognized as “self” by the immune system.

In addition to substituting amino acids at various positions of thedefined nominal epitope, length variants can also be used as analogues.Most typically, additional amino acid residues are added to or removedfrom one, the other, or both ends of a peptide. In some cases,additional terminal residues are removed by proteolysis afteradministration to a subject, regenerating the nominal epitope or ananalogue of the same length before binding with MHC. In other cases, thepeptide of altered length is truly antigenically corss-reactive, asdescribed in Example 20 or exemplified by the relationship betweenMelan-A₂₇₋₃₅ and Melan-A₂₆₋₃₅. It is to be understood that individualembodiments specifically including or excluding any particular aminoacid at any of these additional terminal positions (in some placesherein termed P0 and PΩ+1) are within the scope of the inventiondisclosed herein. When an analogue is said to consist essentially of asequence it is to be understood that such short length variants that areeither trimmed or that retain cross-reactivity are intended. In someembodiments, insertions of smaller amino acids (e.g., glycine, alanine,or serine), especially between the anchor positions, can behavesimilarly to changes in TCR-interacting residues.

1. N-Terminal Proximal Primary Anchor Modification (P2)

The N-terminal primary anchor is the second N-terminal amino acid of thepeptide and is the N-terminal proximal primary anchor. It is primarilyinvolved in the interaction with the MHC molecule and substitutions canresult in improved binding and stability. However, it may be secondarilyinvolved in TCR interactions also. Thus, substitutions at this site canresult in a peptide with improved interaction with MHC molecules as wellas improved interaction with the TCR.

The alanine found at this position in the wild-type sequence isgenerally believed to be non-preferred for the interaction with the MHCmolecule. Thus, preferred embodiments of the analogs have a substitutionat this position. In one embodiment, the original Ala 42 found in thewild-type sequence can be substituted with a more hydrophobic aminoacid. Any more hydrophobic amino acid can be used including any that isavailable or known to one of skill in the art, including standard aminoacids and non-standard amino acids. In a further embodiment, theoriginal Ala 42 is substituted with a more hydrophobic amino acid alsopossessing a bulky side chain. Examples of more hydrophobic amino acidsinclude, but are not limited to: Leu, Val, Ile, Met, α-aminobutyricacid, Norleucine and Norvaline. Table 1 is a summary of the N-terminalproximal primary anchor modifications and the results for each. TABLE 1N-TERMINAL PROXIMAL PRIMARY ANCHOR MODIFICATION Cross-reactivityPredictive Relative and fct avidity Scores Half-maximal affinityStability (native to Catergory Peptide name Sequence SEQ ID NO. (R/NIH)Binding (mM) (1/RA) (T½) (Hrs) analogs)* Native peptide SSX2 41-49KASEKIFYV 1 22/1017  14.64 1.0 11 1 N-terminal SSX2 41-49 (A42L)KLSEKIFYV 102 28/73228 8.89 1.6 19 0.03 Primary Anchor SSX2 41-49 (A42V)KVSEKIFYV 103 22/6407  5.2 2.8 20 0.03 SSX2 41-49 (A42I) KISEKIFYV 10426/10068 8.8 1.7 22.5 3 SSX2 41-49 (A42M) KMSEKIFYV 105 26/52887 8.8 1.722.5 0.1 SSX2 41-49 (A42(D-Ala)) K(D-Ala)SEKIFYV 106 NA N/B N/B N/B 10SSX2 41-49 (A42(D-Leu)) K(D-Leu)SEKIFYV 107 NA N/B N/B N/B N/T SSX241-49 (A42(D-Val)) K(D-Val)SEKIFYV 108 NA N/B N/B N/B 3 SSX2 41-49(A42(Nal-1)) KNal-1SEKIFYV 109 NA N/B N/B N/B >10 SSX2 41-49(A42(Nal-2)) KNal-2SEKIFYV 110 NA 13.9 1.1 N/A 3 SSX2 41-49 (A42(Abu))KAbuSEKIFYV 111 NA 7.56 1.9 N/A 0.3 SSX2 41-49 (A42(Nle)) KNleSEKIFYV112 NA 5.82 2.5 24 0.1 SSX2 41-49 (A42(Nva)) KNvaSEKIFYV 113 NA 11.4 1.3N/A 0.1 SSX2 41-49 (A42(Aib)) KAibSEKIFYV 114 NA 18.4 0.8 N/A 3

2. N-Terminal Secondary Anchor Modification (P1)

The N-terminal secondary anchor is the first amino acid at theN-terminus. This residue is Lys 41 and is defined as a secondary anchorresidue in interacting with the HLA-A*0201 molecule. However, it is alsoengaged in the interaction with the T cell receptors to a certaindegree. Therefore, modifications of this position can generate someheteroclitic analogs that are more immunogenic and more suitable for thedevelopment of tumor vaccines. Although the lysine at this position isgenerally considered to be favored, substitutions can result in highlyimproved properties.

Thus, in one embodiment, the original Lys 43 found in the wild-typesequence can be substituted with a more hydrophobic amino acid. Any morehydrophobic amino acid can be used, including any that is available orknown to one of skill in the art, including standard amino acids andnon-standard amino acids. In a further embodiment, the Lys 43 can besubstituted with an aromatic amino acid. Examples of more hydrophobicamino acids include, but are not limited to: Phe, Tyr, Trp, and D-Lys.Table 2 is a summary of N-terminal secondary anchor modifications andthe results for each. TABLE 2 N-TERMINAL SECONDARY ANCHOR MODIFICATIONSCross-reactivity Half- and Predictive maximal Relative fct avidity SEQID Scores Binding affinity Stability (native to Category Peptide nameSequence NO. (R/NIH) (mM) (1/RA) (T½) (Hrs) analogs)* Native SSX2 41-49KASEKIFYV 1 22/1017 14.64 1.0   11 1 N-terminal SSX2 41-49 (K41F)FASEKIFYV 115 23/1336 9.55 1.5 >24 0.3 Secondary Anchor SSX2 41-49(K41W) WASEKIFYV 116 22/1336 27.07 0.5 N/A >10 SSX2 41-49 (K41Y)YASEKIFYV 117 21/1336 8.74 1.7 >24 3 SSX2 41-49(K41(D-Lys))(D-Lys)ASEKIFYV 118 NA N/B N/B N/B >10 SSX2 41-49 (K41(Phg)) PhgASEKIFYV119 NA 5.83 2.5 >24 0.1 SSX2 41-49 (K41(Cha)) ChaASEKIFYV 120 NA N/B N/BN/B >10 SSX2 41-49 (K41(Phe-4F)) Phe(4-F)ASEKIFYV 121 NA 6.72 2.2 >24 3SSX2 41-49 (K41(Phe-4NO2)) Phe(4-NO2)ASEKIFYV 122 NA 12.8 1.1 N/A 3 SSX241-49 (K41(O-methyl O-methyl-TyrASEKIFYV 123 NA 19.5 0.8   20 3 Tyr))SSX2 41-49 (K41(b-(3- b-(3- 124 NA 24.1 0.6 N/A 10 benzothienyl)Ala))benzothienyl)AlaASEKIFYV

3. N-Terminal Primary and Secondary Modifications (P2 and P1)

In one embodiment, both primary and secondary anchor residues weresubstituted to result in improved binding affinity to the HLA molecule.In a further embodiment, the double substitution produced improvedstability of binding to the HLA molecule. In further embodiments, thebinding and/or stability was not improved and may have even beenreduced, but other properties of the molecule were improved, such asactivity or recognition by a tolerized individual. Table 3 is a summaryof N-terminal primary and secondary anchor modifications and the resultsfor each. TABLE 3 N-TERMINAL PRIMARY AND SECONDARY ANCHOR MODIFICATIONHalf- Cross-reactivity Predictive maximal Relative Stability and fctavidity SEQ Scores Binding affinity (T½) (native to Category Peptidename Sequence ID NO. (R/NIH) (mM) (1/RA) (Hrs) analogs)* Native SSX241-49 KASEKIFYV 1 22/1017  14.64 1.0   11 1 N-terminal SSX2 41-49 (K41Y,A42L) YLSEKIFYV 125 29/96243 11.8 1.2 >24 N/T Primary/ Secondary AnchorSSX2 41-49 (K41Y, A42V) YVSEKIFYV 126 23/8421  14.6 1.0 >24 0.1 SSX241-49 (K41Y, A42M) YMSEKIFYV 127 27/69508 25 0.6 >24 3 SSX2 41-49 (K41Y,A42I) YISEKIFYV 128 27/13233 6.5 2.3 N/A 1 SSX2 41-49 (K41F, A42L)FLSEKIFYV 129 28/96243 4.9 3.0 >24 0.3 SSX2 41-49 (K41F, A42V) FVSEKIFYV130 22/8421  4.675 3.1   24 0.1 SSX2 41-49 (K41F, A42M) FMSEKIFYV 13126/69508 6.58 2.2 >24 3 SSX2 41-49 (K41F, A42I) FISEKIFYV 132 26/132335.368 2.7 >24 0.3 SSX2 41-49 (K41W, A42L) WLSEKIFYV 133 27/96243 4.4723.3 >24 0.3 SSX2 41-49 (K41W, A42V) WVSEKIFYV 134 21/8421  4.82 3.0 >241 SSX2 41-49 (K41W, A42M) WMSEKIFYV 135 25/69508 5.13 2.9 >24 1 SSX241-49 (K41W, A42I) WISEKIFYV 136 25/13233 6.98 2.1 >24 0.1 SSX2 41-49(K41(D-Lys), A42L) (D-Lys)LSEKIFYV 137 N/A 2.5 5.9   15 10 SSX2 41-49(K41(D-Lys), A42V) (D-Lys)VSEKIFYV 138 N/A 24.5 0.6 N/A 10

4. N-Terminal Primary/Secondary Anchor and C-Terminal PrimaryModification (P2, P1 and P9)

The C-terminal Val of the wild-type peptide is generally a preferredanchor residue and primarily involved in the interaction with the MHCmolecule. However, substitutions were carried out to identify whichamino acids improve the analogs having primary and secondary N-terminalmodifications. These C-terminal substitutions can be used in the absenceof one or more N-terminal modifications also.

These modifications were shown to improve binding affinity and stabilityand in some cases resulted in analogs with decreased cross-reactivity.Thus, in some embodiments, the substitution to the C-terminus resultedin a peptide with improved binding and/or stability without decreasedcross-reactivity. However, in other embodiments the substitution to theC-terminus resulted in a peptide with improved binding and/or stabilitywith equal or decreased cross-reactivity. Each of the molecules can beof use in certain cases or in certain patients. In one embodiment, thevaline at the C-terminus is substituted with a large aliphatic aminoacid. Table 4 is a summary of N-terminal primary/secondary anchor andC-terminal primary modifications and the results for each. TABLE 4N-TERMINAL PRIMARY/SECONDARY ANCHOR AND C-TERMINAL PRIMARY MODIFICATIONSCross- Half- reactivity and Predictive maximal Relative Stability fctavidity Scores Binding affinity (T½) (native to Category Peptide nameSequence SEQ ID NO. (R/NIH) (□M) (1/RA) (Hrs) analogs)* Native SSX241-49 KASEKIFYV 1 22/1017 14.64 1.0 11 1 N-terminal SSX2 41-49 (K41F,A42V, V49L) FVSEKIFYL 139 22/2586 10.7 1.4 17 >10 Primary/ SecondaryAnchor, C-terminal Primary Anchor SSX2 41-49 (K41F, A42V, V49I)FVSEKIFYI 140 20/1263 9 1.6 24 0.3 SSX2 41-49 (K41F, A42V, V49A)FVSEKIFYA 141 16/601  6.9 2.1 16 1 SSX2 41-49 (K41F, A42V, V49M)FVSEKIFYM 142 16/601  17.8 0.8 22 >10 SSX2 41-49 (K41F, A42V, V49Nle)FVSEKIFY(Nle) 143 N/A 5.59 2.6 >24   >10 SSX2 41-49 (K41F, A42V,FVSEKIFY(Nva) 144 N/A 1.89 7.7 20 0.1 V49Nva) SSX2 41-49 (K41F, A42V,FVSEKIFY(MeVal) 145 N/A 17.9 0.8 22 10 V49MeVal) SSX2 41-49 (K41F, A42V,V49Aib) FVSEKIFY(Aib) 146 N/A N/A N/A N/A >10 SSX2 41-49 (K41F, A42V,FVSEKIFY(Abu) 147 N/A 3.43 4.3 20 1 V49Abu) N-terminal SSX2 41-49 (A42V,V49I) KVSEKIFYI 148 20/961  13.9 1.1 N/A 0.3 Primary Anchor, C-terminalPrimary Anchor SSX2 41-49 (A42L, V49I) KLSEKIFYI 149  26/10984 5.682 2.6N/A 0.03 SSX2 41-49 (A42a, V49v) K(D- 150 N/A N/B N/B N/B >10Ala)SEKIFY(D- Val) C-terminal SSX2 41-49(V49I) KASEKIFYI 151   20/152.5614 1.0 N/A 10 Primary Anchor

5. N-Terminal Primary/Secondary Anchor and Tcr Residues Modification

The TCR sites are generally recognized as residues P4, P6, and P8 andare the primary residues involved in the binding to the TCR. However,other residues may also be involved in the interaction to a lesserextent. In one embodiment, one or more of the sites primarily involvedin TCR interaction can be substituted to increase the interaction.Preferably, these substitutions can generate heteroclitic analogs thatdo not interfere with binding to the MHC molecule, but overcome thetolerance issues of the wild-type peptides. In a further embodiment, atleast one TCR substitution can be included with at least onesubstitution at position P1, P2, and/or P9. In a further embodiment, thesubstitution at any one or more of the P4, P6, and P8 positions can be apolar amino acid. In a further embodiment, the substitution can be anaromatic amino acid at position P8. In a further embodiment, thesubstitution can be an amino acid with a large aliphatic side chain atposition P6. In a further embodiment, the substitution can be an aminoacid which has a larger side chain to preserve the interaction. Table 5is a summary of N-terminal primary/secondary anchor and TCR residuesmodifications and the results for each. TABLE 5 N-TERMINALPRIMARY/SECONDARY ANCHOR AND TCR SITES MODIFICATION Cross- reactivityHalf- and fct SEQ Predictive maximal Relative avidity ID Scores Bindingaffinity Stability (native to Catergory Peptide name Sequence NO.(R/NIH) (mM) (1/RA) (T½) (Hrs) analogs)* Native SSX2 41-49 KASEKIFYV 122/1017 14.64 1.0 11 1 N-terminal SSX2 41-49 (K41F, A42V, E44D)FVSDKIFYV 152 21/8421 13.18 1.1 N/A >10 Primary/ Secondary Anchor, TCRsites SSX2 41-49 (K41F, A42V, E44N) FVSNKIFYV 153 20/2054 8.97 1.6N/A >10 SSX2 41-49 (K41F, A42V, E44S) FVSSKIFYV 154 20/2054 17.5 0.8N/A >10 SSX2 41-49 (K41F, A42V, E44T) FVSTKIFYV 155 20/2054 12.94 1.1N/A >10 SSX2 41-49 (K41F, A42V, E44Q) FVSQKIFYV 156 20/2054 40.8 0.4 N/A10 SSX2 41-49 (K41F, A42V, E44Nle) FVS(Nle)KIFYV 157 N/A 13 1.1 N/A 10SSX2 41-49 (K41F, A42V, E44Nva) FVS(Nva)KIFYV 158 N/A 3.8 3.9 >24   3SSX2 41-49 (K41F, A42V, I46L) FVSEKLFYV 159 22/8421 7.8 1.9 24 3 SSX241-49 (K41F, A42V, I46V) FVSEKVFYV 160 22/8421 N/A N/A 24 1 SSX2 41-49(K41F, A42V, I46M) FVSEKMFYV 161 18/8421 9.2 1.6 22 >10 SSX2 41-49(K41F, A42V, I46Nle) FVSEK(Nle)FYV 162 N/A 12.8 1.1 19 10 SSX2 41-49(K41F, A42V, I46Nva) FVSEK(Nva)FYV 163 N/A 6.21 2.4 >24   1 SSX2 41-49(K41F, A42V, Y48T) FVSEKIFTV 164 24/1531 3.9 3.8 24 >10 SSX2 41-49(K41F, A42V, Y48F) FVSEKIFFV 165 22/8421 8.8 1.7 20 10 SSX2 41-49 (K41F,A42V, Y48S) FVSEKIFSV 166 24/1531 3.8 3.9 20 >10 SSX2 41-49 (K41F, A42V,Y48(Phe-4F)) FVSEKIF(Phe- 167 N/A 10.6 1.4 24 10 4F)V SSX2 41-49 (K41F,A42V, Y48Phg) FVSEKIF(Phg)V 168 N/A 5.85 2.5 >24   >10 SSX2 41-49 (K41F,A42V, I46L, Y48T) FVSEKLFTV 169 24/1531 5.67 2.6 24 >10 SSX2 41-49(K41F, A42V, I46L, Y48S) FVSEKLFSV 170 24/1531 N/A N/A N/A N/TN-terminal SSX2 41-49 (K41F, A42V, I46L, Y48T, V49A) FVSEKLFTA 17118/109  6.3 2.3 12 >10 Primary/ Secondary Anchor, C-terminal PrimaryAnchor, TCR sites SSX2 41-49 (K41F, A42V, I46L, Y48S, V49A) FVSEKLFSA172 18/109  6.2 2.4 N/A >10

6. C-Terminal Amide

In some embodiments, the C-terminal residue can be modified to containan amide in the place of the free carboxylic acid. Thus, for example, ifthe peptide is a 9-mer (nonamer) the P9 residue can be modified. If thepeptide is a 10-mer (decamer) the P10 residue can be modified.Preferably this results in a peptide or analog that has increasedstability in biological media, including but not limited to blood,lymph, and CNS. Preferably, the peptides can retain the other necessaryactivities to result in an analog usable for vaccination or as animmunogen. Table 6 is a summary of C-terminal amide modifications andthe results for each. TABLE 6 C-TERMINAL AMIDE Predictive RelativeCross-reactivity and Scores Half-maximal affinity Stability fct avidity(native to Catergory Peptide name Sequence SEQ ID NO. (R/NIH) Binding(□M) (1/RA) (T½) (Hrs) analogs)* Native SSX2 41-49 KASEKIFYV 1 22/101714.64 1.0 11 1 C-terminal SSX2 41-49-NH2 KASEKIFYV-NH2 173 N/A N/B N/BN/T >10 amide SSX2 41-49-NH2 (A42L) KLSEKIFYV-NH2 174 N/A N/B N/B N/T 3SSX2 41-49-NH2 (A42V) KVSEKIFYV-NH2 175 N/A N/B N/B N/T 10

7. Decamers

The length of typical MHC binding peptides can vary from about 8 toabout 11 amino acids in length. However, most of the previously usedHLA-A*0201 are 9-mers (nonamers) or 10-mers (decamers). Thus, in oneembodiment, the analog can be an analog of the wild-type sequenceSSX-2₄₁₋₅₀. However, because the wild-type 10-mer does not have thecorrect binding motif and showed no immunological activity, a 10-mer wascreated by substituting amino acids at the P10 position and identifyingthe effect of various wild-type and analogs (see FIG. 1B).

8. Remaining Residues

With reference to FIGS. 1A and 1B, any residues can also be substitutedwith conservative amino acids. Conservative substitutions can be pairedwith any of the above substitutions that can produce an effect.Alternatively, conservative substitutions can be specifically atresidues that are not believed to be involved in any of the activitiesat a primary, secondary, or even tertiary level. Such residues includeP3, P5 and P7. For example, the Serine at position P3 can be substitutedwith an alanine or threonine to produce an analog. Typically, suchconservative substitutions do not significantly affect the activity ofthe analog, however, in some embodiments they can increase certainactivities or decrease certain activities.

NY-ESO-1₁₅₇₋₁₆₅ Analogs

Many features regarding a variety of embodiments and aspects of analogdesign are disclosed above, either generally or as applied to the SSX-2epitope. It is to be understood that such disclosure is also applicableto this and subsequent epitopes. Explicit restatement of such disclosurewill be minimized for the sake of brevity.

Embodiments relate to analogs of the MHC class I-restricted T cellepitope NY-ESO-1₁₅₇₋₁₆₅, SLLMWITQC (SEQ ID NO. 25), polypeptidescomprising these analogs that can be processed by pAPC to present theepitope analogs, and nucleic acids that express the analogs. The analogscan have similar or improved immunological properties compared to thewild-type epitope.

One embodiment relates to methods to derivatize and improve analogs ofNY-ESO-1₁₅₇₋₁₆₅, along with specific sequences that encompasssubstitutions. The analogs can contain at least one substitution, butcan have multiple substitutions comprising standard or non-standardamino acids singly or in various combinations. The analogs can result inpeptides with retained or improved properties.

The epitope NY-ESO-1₅₇₋₁₆₅ has been shown to be presented by NY-ESO-1expressing cell lines, by measuring the epitope specific T cell activityagainst such cells (Jaeger, E. et al., J. Exp. Med. 187:265-270, 1998;U.S. Pat. No. 6,274,145, entitled “ISOLATED NUCLEIC ACID MOLECULEENCODING CANCER ASSOCIATED ANTIGEN, THE ANTIGEN ITSELF, AND USESTHEREOF”, each of which is incorporated herein by reference in itsentirety. Methodologies to improve the physico-chemical properties ofthe peptide NY-ESO-1₁₅₇₋₁₆₅ have been described in U.S. Pat. No.6,417,165, entitled “NY-ESO-1-PEPTIDE DERIVATIVES, AND USES THEREOF”,which is incorporated herein by reference in its entirety, and canconsist of replacement of the terminal cysteine with other amino acidsthat preserve or enhance the interaction with MHC and are devoid of thedeleterious property of disulfide C—C bond formation interfering withthe activity. However, sole manipulation of the C terminal cysteineresidue ignores the advantages of optimizing multiple residuesthroughout the peptide for major histocompatibility (MHC) and/or T cellReceptor (TCR) binding. Thus, beyond the practicality of mutating theCys residue, there is considerable opportunity in mutating additionalamino acids throughout the peptide. For example, substitutions can beused to further optimize the binding to MHC and/or TCR in a fashion thatenables more effective application in clinics.

Embodiments relate to families of one or more peptides of 9 or 10 aminoacids in length related by sequence to amino acids 157-165 of the humancancer testis (CT) antigen NY-ESO-1 (NY-ESO-1₁₅₇₋₁₆₅).

Analog Design

The analog is generally an analog of the NY-ESO-1₁₅₇₋₁₆₅, with thesequence SLLMWITQC (SEQ ID NO: 25). Analysis of whether wild-type aminoacids are preferred or non-preferred used previous analyses of otherpeptide-MHC or TCR interactions. For example, the Cysteine at theC-terminus is generally a non-preferred anchor residue because it doesnot produce a strong interaction with the HLA molecule and, thus, it washighly preferred to substitute this residue. However, although theSerine at position P1 is generally preferred, it was found thatsubstituting an aromatic could produce a peptide with improvedproperties. Further the Leucine at position P2 is generally acceptable,but substituting a hydrophobic and/or bulky amino acid resulted in apeptide with improved properties. The residues are primarily involved inthe interaction with the TCR (P4, P6 and P8) showed a preferencegenerally for some polarity, and in the case of P8 an aromatic generallyproduced peptides with favorable properties.

One preferred embodiment relates to an analog that has a substitution atthe P2 position. In one such embodiment, the substitution can be ahydrophobic residue. In a further embodiment, the substitution can be abulky hydrophobic residue. In another embodiment, the residue at P1 canbe substituted with a more hydrophobic residue. In a further embodiment,residues P1 and P2 can be both substituted with more hydrophobicresidues. In further embodiments, at least one residue at P1, P2, and P9can be substituted. In a further embodiment, at least two residues atP1, P2 and P9 can be substituted. In a further embodiment, at least tworesidues at P1, P2, P9, P4, and P6 can be substituted, including one ormore residues involved in TCR binding. In a further embodiment, theresidue at P8 can be substituted with an aromatic residue. Examples ofthe following substitutions are shown in FIGS. 13A-13C.

1. N-Terminal Proximal Primary Anchor Modification (P2)

The N-terminal primary anchor is the second N-terminal amino acid of thepeptide, thus, it is the N-terminal proximal primary anchor. Althoughthe original Leucine 158 is not considered “non-preferred” for bindingto the MHC molecule, substitutions can produce a peptide with improvedbinding. Thus, in one embodiment, the original Leu 158 found in thewild-type sequence can be substituted with a similarly or morehydrophobic amino acid. Any hydrophobic amino acid can be used,including one that is available to or that is known to one of skill inthe art, including standard amino acids and non-standard amino acids. Ina further embodiment, the original Leu 158 can be substituted with amore hydrophobic amino acid also possessing a bulky side chain. Examplesof more hydrophobic amino acids include, but are not limited to: Leu,Val, Ile, Met, α-aminobutyric acid, Norleucine and Norvaline. Further, anaphthal side chain can also be substituted. Preferably, thesubstitution results in improved binding and stability with the HLAmolecule. However, this residue may be secondarily or tertiarilyinvolved in TCR interactions, and substitutions can also result inimproved recognition by the TCR.

2. N-Terminal Secondary Anchor Modification (P1)

The N-terminal secondary anchor is the first amino acid at theN-terminus or P1. This residue is involved in a number of interactions.The residue of Ser 157 was defined as a secondary anchor residue ininteracting with HLA-A*0201 molecule. It is also engaged in theinteraction with the T cell receptors to a certain degree. Therefore,modifications of this position generate some heteroclitic analogs thatare more immunogenic and more suitable for the development of tumorvaccines. Thus, substitutions can result in a variety of improvedqualities.

Although the Serine is not considered “non-preferred,” a number ofsubstitutions can result in improved qualities of the peptide. Thus, inone embodiment, the original Ser 157 found in the wild-type sequence canbe substituted with a more hydrophobic amino acid. Any more hydrophobicamino acid can be used, including one that is available to or that isknown to one of skill in the art, including standard amino acids andnon-standard amino acids. Examples of more hydrophobic amino acidsinclude, but are not limited to: Phe, Tyr, Trp, and D-Lys.

3. N-Terminal Primary and Secondary Modifications (P2 and P1)

In one embodiment, both primary and secondary anchor residues weresubstituted to result in improved binding affinity to the HLA molecule.In a further embodiment, the double substitution produced improvedstability of binding to the HLA molecule. In further embodiments, thebinding and/or stability was not improved and may have even beenreduced, but other properties of the molecule were improved, such asactivity or recognition by a tolerized individual.

4. N-Terminal Primary/Secondary Anchor and C-Terminal PrimaryModification (P2, P1 and P9)

The C-terminal cysteine of the wild-type peptide is generally anon-preferred anchor residue. Because this residue is generallyprimarily involved in the interaction with the MHC molecule, it can bepreferred to substitute residues that result in a stronger interactionwith the MHC molecule. Thus, substitutions were shown to improve bindingaffinity and stability and in some cases resulted in analogs withdecreased cross-reactivity. In some embodiments, the substitution to theC-terminus can result in a peptide with improved binding and/orstability without decreased cross-reactivity. However, in otherembodiments, the substitution to the C-terminus can result in a peptidewith improved binding and/or stability with equal or decreasedcross-reactivity. Because substitution of this residue has beenpreviously shown to provide improved peptides, it can be preferableproduce peptides that are more improved in the interaction with the MHCmolecule as well as other interactions, such as the recognition by theTCR. Thus, in some embodiments, the C-terminal substitution can bepaired with at least one other substitution. Examples of amino acidsubstitutions to the C-terminus include, but are not limited to, valine,lysine, alanine, and isoleucine.

5. N-Terminal Primary/Secondary Anchor and Tcr Residue Modifications

The primary residues involved in the interaction with the TCR aregenerally recognized as residues P4, P6, and P8. However, other residuesmay also be involved in the interaction to a lesser extent. In oneembodiment, one or more of the sites primarily involved in TCRinteraction can be substituted to result in an improved interaction.Preferably, these substitutions generate heteroclitic analogs that donot interfere with binding to the MHC molecule, but overcome thetolerance issues of the wild-type peptides. In one embodiment, at leastone TCR substitution can be included with at least one substitution atposition P1, P2, and/or P9. In one embodiment, amino acids with somepolarity can be substituted at P4, P6, and P8. In a further embodiment,aromatic amino acids can be substituted at the P8 position.

6. C-Terminal Amide

In some embodiments, the C-terminal residue can be modified to containan amide in the place of the free carboxylic acid. Thus, for example ifthe peptide is a 9-mer (nonamer) the P9 residue can be modified. If thepeptide is a 10-mer (decamer) the P10 residue can be modified.Preferably, this results in a peptide or analog that has increasedstability in biological media, including but not limited to blood,lymph, and CNS. Preferably, the peptides retain their other activitiesto result in an analog usable for vaccination or as an immunogen.

7. Decamers

The length of typical MHC binding peptides varies from about 8 to about11 amino acids in length. However, most of the previously usedHLA-A*0201 are 9-mers (nonamers) or 10-mers (decamers). Thus, in oneembodiment, the analog can be a 10-mer of the wild-type sequenceNY-ESO-1₁₅₇₋₁₆₆. However, because the wild-type 10-mer does not have thecorrect binding motif and showed no immunological activity, a 10-mer wascreated by substituting amino acids at the P10 position and identifyingthe effect of various modifications (see FIGS. 13A-13C). In oneembodiment, the residues that were added or substituted for thewild-type at the C-terminus can be selected from the group consisting ofnorvaline, leucine, isoleucine, valine, and alanine.

8. Remaining Residues

With reference to FIGS. 13A and 13C, any residues can also besubstituted with conservative amino acids. Conservative substitutionscan be paired with any of the above substitutions that can produce aneffect. Alternatively, conservative substitutions can be specifically atresidues that are not believed to be involved in any of the activitiesat a primary, secondary, or even tertiary level. Such residues caninclude P3, P5 and/or P7. Conservative substitutions are known to thoseof skill in the art, but, for example, the Leucine at position P3 can besubstituted with an alanine or threonine to produce an analog.Typically, such conservative substitutions do not significantly affectthe activity of the analog. However, in some embodiments they mayincrease certain activities or decrease certain activities. Because ofthe known interactions, it is unlikely that such conservativesubstitutions will have a significant effect on any of the activities.

PSMA₂₈₈₋₂₉₇ Analogs

Many features regarding the variety of embodiments and aspects of analogdesign are disclosed above, either generally or as applied to particularepitopes. It is to be understood that such disclosure is also applicableto this and subsequent epitopes. Explicit restatement of such disclosurewill be minimized for the sake of brevity.

Some embodiments relate to analogs of the MHC class I-restricted T cellepitope PSMA₂₈₈₋₂₉₇, GLPSIPVHPI (SEQ ID NO. 42), polypeptides comprisingthese analogs that can be processed by pAPC to present the epitopeanalogs, and nucleic acids that express the analogs. The analogs canhave similar or improved immunological properties compared to thewild-type epitope. Evidence validating the presentation of this epitopeby human cancer cells is presented in Example 32 below.

One embodiment relates to methods to derivatize and improve analogs ofPSMA₂₈₈₋₂₉₇, along with specific sequences that encompass substitutions.The analogs can contain at least one substitution, but can have multiplesubstitutions comprising standard or non-standard amino acids singly orin various combinations. The analogs can result in peptides withretained or improved properties.

Embodiments relate to families of one or more peptides of 9 or 10 aminoacids in length related by sequence to amino acids 288-297 of the humanPSMA.

Analog Design

In some embodiments, the PSMA₂₈₈₋₂₉₇ analog can contain substitutions ofthe sequence GLPSIPVHPI (SEQ ID NO. 42). Reference to binding motifdata, such as presented in table 7 in example 2 below, indicates thatthe P2 anchor residue can make the largest individual contribution toaffinity of any position in an A2.1-restricted epitope. In this case,the amino acid at the P2 position is the optimally preferred leucine.The PΩ anchor residue, isoleucine, is favorable. In vitro bindingstudies using the T2 cell assay system (not shown) have indicated thatthe native peptide has generally superior binding characteristics,particularly as compared to the SSX-2 and NY-ESO-1 epitopes. The epitopeexhibited significant binding at relatively low concentrations, althoughthis was paired with a relatively shallow rise toward saturation. Thewild-type epitope can be improved. Analyses such as those represented bytables 7 and 8 are averages and the behavior of a given residue in aparticular sequence may diverge from the average. Consistent with thefavorable results obtained with Nle and Nva for the SSX-2 and NY-ESO-1epitopes discussed above, Nle and Nva also can be successfully used forthe instant PSMA epitope. Finally, even similar binding characteristics,if paired with alterations that help circumvent whatever tolerance tothe epitope may exist, can increase the effective immunogenicity of thepeptide. In the transgenic mouse model, the native peptide is poorlyimmunogenic (see Example 35 for instance) which may reflect tolerance tothe epitope; the region of PSMA from which this epitope is derived isidentical between mouse and human PSMA.

1. N-Terminus Proximal Primary Anchor Modification (P2)

As noted above, although the native residue at the P2 position of thisepitope is generally the optimal residue among genetically encoded aminoacids. The effect of substituting other preferred or bulky hydrophobicresidues were examined for potential improvement of binding, tolerancebreaking and cross-reactive immunity. Exemplary substitutions caninclude Met, Ile, Gln, Val, Nva, Nle, and aminobutyric acid (Abu).

2. N-Terminal Secondary Anchor Modification (P1)

The N-terminal secondary anchor is the first amino acid at theN-terminus. The native Gly is only marginally preferred at thisposition. Various observations (see tables 7 and 8 for example) showthat amino acids with potential to improve the epitope include Ala, Ser,Abu and sarkosine (Sar, that is, N-methylglycine).

3. C-Terminal Primary Anchor Modification (PΩ)

The native Ile at this position is generally a preferred but not optimalresidue. Substitution at this position can improve binding. Exemplarysubstitutions can include Val, Leu, Nva, and Nle.

4. Secondary Anchors and Tcr Exploration

The penultimate position (PΩ-1) can serve both as a secondary anchor anda TCR interacting position. Substitution of Ala, Leu, Ser, or Thr canhave a primary effect on TCR interaction, though it can also contributeto improved binding. P3 is another position that can effect both bindingand immunogenicity. Substitution of Trp at this position can improveboth.

Further embodiments relate to combinations of substitutions at multiplepositions in order to combine, synergize, and counteract the variouseffects obtained with the single substitutions.

PRAME₄₂₅₋₄₃₃ Analogs

Many features regarding a variety of embodiments and aspects of analogdesign are disclosed above, either generally or as applied to particularepitopes. It is to be understood that such disclosure is also applicableto this and subsequent epitopes. Explicit restatement of such disclosurewill be minimized for the sake of brevity.

Embodiments include analogs of the MHC class I-restricted T cell epitopePRAME₄₂₅₋₄₃₃, SLLQHLIGL (SEQ ID NO. 71), polypeptides comprising theseanalogs that can be processed by pAPC to present the epitope analogs,and nucleic acids that express the analogs. The analogs can have similaror improved immunological properties compared to the wild-type epitope.Evidence validating the presentation of this epitope by human cancercells is presented in Example 39 below.

One embodiment relates to methods to derivatize and improve analogs ofPRAME₄₂₅₋₄₃₃, along with specific sequences that encompasssubstitutions. The analogs can contain at least one substitution, butcan have multiple substitutions comprising standard or non-standardamino acids singly or in various combinations. The analogs can result inpeptides with retained or improved properties.

Some embodiments relate to families of one or more peptides of 9 or 10amino acids in length related by sequence to amino acids 425-433 of thehuman PRAME sequence.

Analog Design

Some embodiments relate to analogs of the PRAME₄₂₅₋₄₃₃ which can containsubstitutions of the sequence SLLQHLIGL (SEQ ID NO. 71). Reference tobinding motif data, such as those presented in Table 7 in Example 2below, indicates that the P2 anchor residue can make the largestindividual contribution to affinity of any position in anA2.1-restricted epitope. In this case, the amino acid at the P2 positionis the optimally preferred leucine. The PΩ anchor residue, leucine, isfavorable, though not as strongly preferred, nor is the wild type PΩresidue necessarily the most preferred for that position. Analyses suchas those reported in Tables 7 and 8 are averages and the behavior of agiven residue in a particular sequence can diverge from the average.Consistent with the favorable results obtained with Nle and Nva for theother epitopes, similar improvements can be obtained substituting Nleand Nva with this sequence. Finally, even similar bindingcharacteristics, if paired with alterations that help circumventwhatever tolerance to the epitope may exist, can increase the effectiveimmunogenicity of the peptide.

The rationale for various substitutions has been set forth above. Theparticular substitutions investigated for the PRAME₄₂₅₋₄₃₃ epitopefollow the same logic and are disclosed in the Examples 40-48 and FIGS.25-27. Substitutions were made at the primary anchor positions P2 and PΩ(P9), the secondary anchor positions P1 and PΩ-1 (P8). Substitutionswere also made in the TCR interacting positions (in addition tosecondary anchor positions) P3 and P6. Selected substitutions haveimpact on binding and/or stability of MHC class I—peptide complexes; akey feature in determining the immunological properties of peptides. Inaddition, due to T cell repertoire considerations and to circumventmechanisms responsible for the limited immunity to native epitopes,substitutions that retain the capability of analogs to interact with Tcell receptors recognizing native peptides can be of practical value.

EXAMPLES

The following examples provide analogs and methods of identifyinganalogs. The analogs can be used, for example, as immunogens, vaccines,and/or for the treatment of a variety of cancers. The analogs wereproduced as in Example 1. SSX-2₄₁₋₄₉ analogs were identified as shown inExample 2 and are listed in Example 3. The analogs were tested forimproved properties as shown in Examples 4-21. The testing ofNY-ESO-1₁₅₇₋₁₆₅ analogs for improved properties is presented in Examples22-30. The testing of PSMA analogues for improved properties ispresented in Examples 33-38. The testing of PRAME₄₂₅₋₄₃₃ analogues forimproved properties is presented in Examples 40-48.

Example 1 Peptide Synthesis, Purification and Characterization

Peptides were synthesized on either a Symphony multiple peptidesynthesizer (PTI technologies, MA) or an ABI 433A peptide synthesizer(Applied Biosystems, Foster City, Calif.) at 0.05-0.1 mmole scale usingstandard Fmoc solid phase chemistry. C-terminal free acid peptides weresynthesized using pre-load PEG-PS resins (on Symphony) or Wang resin (onABI). C-terminal amidated peptides were synthesized on Fmoc-PAL-PEG-PSresin. All resins were purchased from Applied Biosystems (Foster City,Calif.). The Fmoc-amino acids used in peptide syntheses were purchasedfrom Novabiochem (San Diego, Calif.) and AnaSpec (San Jose, Calif.).Post-synthesis cleavage was carried on by the standard protocol.

Peptide purification was carried out on either semi-preparative HPLCcolumns or SPE cartridges (Phenomenex, Torrance, Calif.). The purity ofall peptides was >90%. The identity of each peptide was verified byMaldi-TOF MS (Voyager DE, Applied Biosystems) and analytical HPLCs(Varian or Shimazu) using a Synergi C12 column (Phenomenex, Torrance,Calif.).

Example 2 De Novo Designed SSX-2₄₁₋₄₉ Analogs

Structural modification of a moderately antigenic peptide canconsiderably improve peptide-MHC binding, CTL recognition, and/orimmunogenicity. General guidelines regarding how to modify a wild-typeepitope in order to achieve a peptide analog with enhanced potency areknown in the art. An appreciated strategy is to optimize the residues atthe so-called anchor positions for binding to the particular MHCmolecule at issue. In the case of HLA-A2, a marked preference forhydrophobic residues at the P2 and PΩ positions has been observed,particularly L and M at P2, and V at PΩ. (PΩ denotes the C-terminalresidue of the epitope. For HLA-A2, that is P9 or P10 depending on thelength of the peptide.) Replacing the P1 position with aromaticresidues, such as F, Y and W can also be advantageous. TABLE 7Coefficients used by the BIMAS algorithm (Algorithm available byhypertext transfer protocol: //bimas.cit.nih.gov/molbio/hla_bind/) 9-merCoefficient Table for HLA_A_0201 Amino Acid Position Type 1st 2nd 3rd4th 5th 6th 7th 8th 9th A 1.000 1.000 1.000 1.000 1.000 1.000 1.0001.000 1.000 C 1.000 0.470 1.000 1.000 1.000 1.000 1.000 1.000 1.000 D0.075 0.100 0.400 4.100 1.000 1.000 0.490 1.000 0.003 E 0.075 1.4000.064 4.100 1.000 1.000 0.490 1.000 0.003 F 4.600 0.050 3.700 1.0003.800 1.900 5.800 5.500 0.015 G 1.000 0.470 1.000 1.000 1.000 1.0000.130 1.000 0.015 H 0.034 0.050 1.000 1.000 1.000 1.000 1.000 1.0000.015 I 1.700 9.900 1.000 1.000 1.000 2.300 1.000 0.410 2.100 K 3.5000.100 0.035 1.000 1.000 1.000 1.000 1.000 0.003 L 1.700 72.000 3.7001.000 1.000 2.300 1.000 1.000 4.300 M 1.700 52.000 3.700 1.000 1.0002.300 1.000 1.000 1.000 N 1.000 0.470 1.000 1.000 1.000 1.000 1.0001.000 0.015 P 0.022 0.470 1.000 1.000 1.000 1.000 1.000 1.000 0.003 Q1.000 7.300 1.000 1.000 1.000 1.000 1.000 1.000 0.003 R 1.000 0.0100.076 1.000 1.000 1.000 0.200 1.000 0.003 S 1.000 0.470 1.000 1.0001.000 1.000 1.000 1.000 0.015 T 1.000 1.000 1.000 1.000 1.000 1.0001.000 1.000 1.500 V 1.700 6.300 1.000 1.000 1.000 2.300 1.000 0.41014.000 W 4.600 0.010 8.300 1.000 1.000 1.700 7.500 5.500 0.015 Y 4.6000.010 3.200 1.000 1.000 1.500 1.000 5.500 0.015 final 0.069 constant

TABLE 8 Scoring Pattern for HLA-A*0201 used by the SYFPEITHI Algorithm(9-mers) (Algorithm available by hypertext transfer protocol://syfpeithi.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm) AA P1 P2P3 P4 P5 P6 P7 P8 P9 A 2 6 2 0 0 0 2 1 6 C 0 0 0 0 0 0 0 0 0 D −1 0 0 10 0 0 0 0 E −1 0 −1 2 0 0 0 2 0 F 1 0 1 −1 1 0 0 0 0 G 1 0 0 2 2 0 0 1 0H 0 0 0 0 0 0 1 0 0 I 2 8 2 0 0 6 0 0 8 K 1 0 −1 0 1 0 −1 2 0 L 2 10 2 01 6 1 0 10 M 0 8 1 0 0 0 0 0 6 N 0 0 1 0 0 0 1 0 0 P 0 0 0 2 1 0 1 0 0 Q0 0 0 0 0 0 0 0 0 R 0 0 0 0 0 0 0 0 0 S 2 0 0 0 0 0 0 2 0 T 0 6 −1 0 0 20 2 6 V 1 6 0 0 0 6 2 0 10 W 0 0 1 0 0 0 0 0 0 X 0 0 0 0 0 0 0 0 0 Y 2 01 −1 1 0 1 0 0

Adapted from: Rammensee, Bachmann, Stevanovic: MHC ligands and peptidemotifs. Landes Bioscience 1997

Example 3

The following analogs were produced using the predictions in Example 1.TABLE 9 SEQ ID Catergory Number Peptide name Sequence wild-type 1 SX-241-49 KASEKIFYV N-terminal 102 SSX-2 41-49 (A42L) KLSEKIFYV Primary 103SSX-2 41-49 (A42V) KVSEKIFYV Anchor 104 SSX-2 41-49 (A42I) KISEKIFYV 105SSX-2 41-49 (A42M) KMSEKIFYV 106 SSX-2 41-49 (A42(D-Ala))K(D-Ala)SEKIFYV 107 SSX-2 41-49 (A42(D-Leu)) K(D-Leu)SEKIFYV 108 SSX-241-49 (A42(D-Val)) K(D-Val)SEKIFYV 109 SSX-2 41-49 (A42(Nal-1))KNal-1SEKIFYV 110 SSX-2 41-49 (A42(Nal-2)) KNal-2SEKIFYV 111 SSX-2 41-49(A42(Abu)) KAbuSEKIFYV 112 SSX-2 41-49 (A42(Nle)) KNleSEKIFYV 113 SSX-241-49 (A42(Nva)) KNvaSEKIFYV 114 SSX-2 41-49 (A42(Aib)) KAibSEKIFYVN-terminal 115 SSX-2 41-49 (K41F) FASEKIFYV Secondary 116 SSX-2 41-49(K41W) WASEKIFYV Anchor 117 SSX-2 41-49 (K41Y) YASEKIFYV 118 SSX-2 41-49(K41(D-Lys)) (D-Lys)ASEKIFYV 119 SSX-2 41-49 (K41(Phg)) PhgASEKIFYV 120SSX-2 41-49 (K41(Cha)) ChaASEKIFYV 121 SSX-2 41-49 (K41(Phe-4F))Phe(4-F)ASEKIFYV 122 SSX-2 41-49 (K41(Phe-4NO2)) Phe(4-NO ₂)ASEKIFYV 123SSX-2 41-49 (K41(O-methyl Tyr)) O-methyl-TyrASEKIFYV 124 SSX-2 41-49(K41(β-(3-benzothienyl)Ala)) β-(3-benzothienyl)AlaASEKIFYV N-terminal125 SSX-2 41-49 (K41Y, A42L) YLSEKIFYV Primary/ 126 SSX-2 41-49 (K41Y,A42V) YVSEKIFYV Secondary 127 SSX-2 41-49 (K41Y, A42M) YMSEKIFYV Anchor128 SSX-2 41-49 (K41Y, A42I) YISEKIFYV 129 SSX-2 41-49 (K41F, A42L)FLSEKIFYV 130 SSX-2 41-49 (K41F, A42V) FVSEKIFYV 131 SSX-2 41-49 (K41F,A42M) FMSEKIFYV 132 SSX-2 41-49 (K41F, A42I) FISEKIFYV 133 SSX-2 41-49(K41W, A42L) WLSEKIFYV 134 SSX-2 41-49 (K41W, A42V) WVSEKIFYV 135 SSX-241-49 (K41W, A42M) WMSEKIFYV 136 SSX-2 41-49 (K41W, A42I) WISEKIFYV 137SSX-2 41-49 (K41(D-Lys), A42L) (D-Lys)LSEKIFYV 138 SSX-2 41-49(K41(D-Lys), A42V) (D-Lys)VSEKIFYV N-terminal 139 SSX-2 41-49 (K41F,A42V, V49L) FVSEKIFYL Primary/ 140 SSX-2 41-49 (K41F, A42V, V49I)FVSEKIFYI Secondary 141 SSX-2 41-49 (K41F, A42V, V49A) FVSEKIFYA Anchor,142 SSX-2 41-49 (K41F, A42V, V49M) FVSEKIFYM C-terminal 143 SSX-2 41-49(K41F, A42V, V49Nle) FVSEKIFY(Nle) Primary 144 SSX-2 41-49 (K41F, A42V,V49Nva) FVSEKIFY(Nva) Anchor 145 SSX-2 41-49 (K41F, A42V, V49MeVal)FVSEKIFY(MeVal) 176 SSX-2 41-49 (K41F, A42V, V49MeLeu) FVSEKIFY(MeLeu)146 SSX-2 41-49 (K41F, A42V, V49Aib) FVSEKIFY(Aib) 147 SSX-2 41-49(K41F, A42V, V49Abu) FVSEKIFY(Abu) N-terminal 152 SSX-2 41-49 (K41F,A42V, E44D) FVSDKIFYV Primary/ 153 SSX-2 41-49 (K41F, A42V, E44N)FVSNKIFYV Secondary 154 SSX-2 41-49 (K41F, A42V, E44S) FVSSKIFYV Anchor,155 SSX-2 41-49 (K41F, A42V, E44T) FVSTKIFYV TCR sites 156 SSX-2 41-49(K41F, A42V, E44Q) FVSQKIFYV 157 SSX-2 41-49 (K41F, A42V, E44(Nle))FVS(Nle)KIFYV 158 SSX-2 41-49 (K41F, A42V, E44(Nva)) FVS(Nva)KIFYV 159SSX-2 41-49 (K41F, A42V, I46L) FVSEKLFYV 160 SSX-2 41-49 (K41F, A42V,I46V) FVSEKVFYV 161 SSX-2 41-49 (K41F, A42V, I46M) FVSEKMFYV 162 SSX-241-49 (K41F, A42V, I46(Nle)) FVSEK(Nle)FYV 163 SSX-2 41-49 (K41F, A42V,I46(Nva)) FVSEK(Nva)FYV 164 SSX-2 41-49 (K41F, A42V, Y48T) FVSEKIFTV 165SSX-2 41-49 (K41F, A42V, Y48F) FVSEKIFFV 166 SSX-2 41-49 (K41F, A42V,Y48S) FVSEKIFSV 167 SSX-2 41-49 (K41F, A42V, Y48(Phe-4F))FVSEKIF(Phe4-F)V 168 SSX-2 41-49 (K41F, A42V, Y48(Phg)) FVSEKIF(Phg)V169 SSX-2 41-49 (K41F, A42V, I46L,Y48T) FVSEKLFTV 170 SSX-2 41-49 (K41F,A42V, I46L,Y48S) FVSEKLFSV N-terminal 171 SSX-2 41-49 (K41F, A42V,I46L,Y48T,V49A) FVSEKLFTA Primary/ 172 SSX-2 41-49 (K41F, A42V,I46L,Y48S,V49A) FVSEKLFSA Secondary Anchor, C-Terminal Primary Anchor,TCR sites N-terminal 148 SSX-2 41-49 (A42V, V49I) KVSEKIFYI Primary 149SSX-2 41-49 (A42L, V49I) KLSEKIFYI Anchor, 150 SSX-2 41-49 (A42(D-Ala),V49(D-Val)) (D-Ala)SEKIFY(D-Val) C-terminal SSX-2 41-49 (A42(D-Leu),V49(D-Val)) (D-Leu)SEKIFY(D-Val) Primary SSX-2 41-49 (A42(D-Val),V49(D-Val)) (D-Val)SEKIFY(D-Val) Anchor C-terminal 151 SSX-2 41-49(V491) KASEKIFYI Primary Anchor C-terminal 173 SSX-2 41-49-NH2KASEKIFYV-NH2 amide 174 SSX-2 41-49-NH2 (A42L) KLSEKIFYV-NH2 175 SSX-241-49-NH2 (A42V) KVSEKIFYV-NH2 Decamers 177 SSX-2 41-50 KASEKIFYVY 178SSX-2 41-50 (Y50I) KASEKIFYVI 179 SSX-2 41-50 (Y50L) KASEKIFYVL 180SSX-2 41-50 (Y50V) KASEKIFYVV 181 SSX-2 41-50 (Y50(Nle)) KASEKIFYV(Nle)182 SSX-2 41-50 (Y50(Nva)) KASEKIFYV(Nva) 183 SSX-2 41-50 (A42V,Y50I)KVSEKIFYVI 184 SSX-2 41-50 (A42L,Y50I) KLSEKIFYVI 185 SSX-2 41-50(A42V,Y50L) KVSEKIFYVL 186 SSX-2 41-50 (A42L,Y50L) KLSEKIFYVL 187 SSX-241-50 (A42V,Y50V) KVSEKIFYVV 188 SSX-2 41-50 (A42L,Y50V) KLSEKIFYVV 189SSX-2 41-50 (A42V,Y50(Nle)) KVSEKIFYV(Nle) 190 SSX-2 41-50(A42L,Y50(Nle)) KLSEKIFYV(Nle) 191 SSX-2 41-50 (A42V,Y50(Nva))KVSEKIFYV(Nva) 192 SSX-2 41-50 (A42L,Y50(Nva)) KLSEKIFYV(Nva) 193 SSX-241-50 (A42V,V49I,Y50I) KVSEKIFYII 194 SSX-2 41-50 (A42L,V49I,Y50I)KLSEKIFYII 195 SSX-2 41-50 (V491,Y501) KASEKIFYII

Abbreviations for non-standard amino acids are as follows: Nle,norleucine; Nva, norvaline; Phg, phenylglycine; Phe(4-F),4-fluorophenylalanine; Phe(4-NO₂), 4-nitrophenylalanine; Abu,α-aminobutyric acid; Aib, α-aminoisobutyric acid; MeLeu, methyl-leucine;MeVal, methylvaline; β-(3-benzothienyl)Ala, β-(3-benzothienyl)-alanine;O-methyl-Tyr, O-methyltyorosine; Cha, cyclohexylalanine; Nal-1,β-(1-napthyl)-alanine; Nal-2, β-(2-napthyl)-alanine; —NH2 indicates thatthe carboxy terminus has been modified to the amide.

Examples 4-21 Testing of SSX-2₄₁₋₄₉ Analogs

The analogs produced in Example 3 were tested for activity, such asbinding and biological effect as follows in Examples 4-21:

Example 4 Peptide Binding Using T2 Cells

The affinity of peptide analogs and the wild-type epitope to HLA-A*0201was evaluated using a T2 cell based assay (Regner M, et al., Exp ClinImmunogenet. 1996; 13(1):30-5; which is hereby incorporated by referencein its entirety).

For the binding assay, in brief, T2 cells that lack expression of TAPand thus do not assemble stable MHC class I on the cell surface, werepulsed with different concentrations of peptides (controls or analogs)overnight at 37° C., washed extensively, stained with fluorescentlytagged antibody recognizing MHC class I (A2 allele) and run through aFacsScan analyzer. The difference between the MFI (mean fluorescenceintensity) corresponding to a given concentration of analog and thenegative control (non-MHC binder) is a function of how many stabilizedcomplexes between MHC and peptide are displayed on the surface of T2cells. Thus, at limiting concentrations of peptide, it is a measurementof K_(on) mostly and at saturation levels of peptide it is a measurementof both K_(on) and K_(off). The binding was quantified by two factorsthat are mathematically related: half maximal binding (the peptideconcentration giving 50% of the signal corresponding to saturation) andrelative affinity (1/RA). Relative affinity, RA, is binding normalizedto a reference (wild-type peptide); for example, the ratio between halfmaximal binding of control relative to peptide analog. The higher the1/RA index and the lower the half maximal binding, the higher the K_(on)of the interaction between the analog and the MHC.

Fifty three analogs identified with these binding parameters weredetermined to be improved relative to the wild-type peptide. Theseimproved binders carry one, two, three or multiple substitutions(including standard and/or non-standard amino acids) involving positionsthat are known to participate in the interaction with MHC and/or TCR.However, the overall effect on MHC binding was dependent on themodification. Such peptide analogs can be useful in therapeuticcompositions or as a platform to further derive therapeuticcompositions.

Example 5 Peptide Stability Using T2 Cells

Peptide stability (K_(off)) on MHC generally cannot be solely inferredfrom binding (K_(on)). In addition to binding, the stability of peptideson MHC class I is notoriously important with regard to the immunologicalproperties of such peptides, as the activation of T cells depends on theduration of “signal 1” (MHC peptide complex interaction with T cellreceptor). For the stability assay, in brief, T2 cells that lackexpression of TAP, and thus do not assemble stable MHC class I on thecell surface, were pulsed with a concentration of peptide (controls oranalogs) known to achieve maximal loading of MHC class I (“saturation”)overnight at 37° C., washed extensively, and chased for differentintervals in the presence of emetine, which blocks endogenous proteinsynthesis. After extensive washing, the cells were stained withfluorescently tagged antibody recognizing MHC class I (A2 allele) andrun through a FacsScan analyzer. The difference between the MFI (meanfluorescence intensity) corresponding to a given concentration of analogand the negative control (non-MHC binder) is a function of how manystabilized complexes between MHC and peptide are displayed on thesurface of T2 cells. The decay of the signal over time wasmathematically expressed as stability index 50% relative to the bindingat 0 hours (at the beginning of the chase interval).

Such improved analogs can carry one, two, three or multiplesubstitutions (including standard and/or non-standard amino acids)involving positions that are known to participate in the interactionwith MHC and/or TCR, with an overall effect on MHC stability that isdependent on the modification. Such peptide analogs can be useful intherapeutic compositions or as a platform to further derive therapeuticcompositions. Forty three of the analogs had increased stabilityrelative to the natural peptide.

The analogs that showed both improved binding and stability are usefulin improved compositions or as a platform to generate improvedcompositions of therapeutic benefit.

Example 6 Evaluation of Immunologic Properties of Analogs:Cross-Reactivity and Functional Avidity

The immunologic properties of peptides can be described as a function ofbinding to MHC molecules (K_(on) and K_(off)) and TCR (affinity ofinteraction between TCR and MHC-peptide complexes). Modifications ofprimary MHC anchor residues generally have a significant degree ofpredictability in regard to overall impact on binding to MHC molecules.

Modifications of secondary MHC anchor residues can impact the affinityof interaction of the MHC-peptide complex to TCR along with the K_(on)and K_(off) relative to peptide-MHC interaction.

A methodology was devised to allow rapid and rational screening ofpeptide analogs in a fashion coherent with proposed methods of use andmodeling the overall immunologic properties (K_(on) and K_(off) relativeto MHC interaction and TCR binding properties in an integrated fashion).In some instances, the method included generating T cell lines against anatural (non-mutated) epitope (SSX-2₄₁₋₄₉), and using an immunizationstrategy potent enough to generate a useful response in transgenic micecarrying human MHC (such as the A2 allele). Peptide analogs wereinterrogated ex vivo in the presence of competent APCs and thefunctional impact of T cells specific for natural (non-mutated) epitopesmeasured. The evaluation was done at various concentrations of analog,because the expected effect was biphasic in the case of cross reactivepeptides (activating at limited concentrations and inhibiting at higherconcentrations, due to antigen-induced cell death, AICD). Measurement ofthe following three parameters were used to define basic and usefulcharacteristics of peptide analogs:

-   -   1. Minimal required concentration of peptide analog to trigger        effects indicative of T cell activation (e.g. cytokine        production);    -   2. Maximal (peak value) effect (e.g. cytokine production) at any        analog concentration;    -   3. Analog concentration at peak value of activating effect        (e.g., cytokine concentration)

For example, analogs that result in reduced values associated withparameters #1 and 3 but increased #2, can be useful. Use of naturalepitope and unrelated non-cross reactive peptides as references isvaluable in identifying classes of analogs of potential value. Analogsthat display properties quantitatively comparable to or even modestlyattenuated from those of natural epitopes are still deemed useful inlight of the fact that while they retain cross-reactivity, they maydisplay immunologic properties that are distinct from those of thenatural peptide—for example, lower propensity to induce AICD or abilityto break tolerance or restore responsiveness in vivo.

Some advantages of this screening strategy include the practicality andrapidity, use of more relevant polyclonal T cell lines instead ofpotentially biased T cell clones as a read out, and the composite value,integrating parameters such as K_(on), K_(off) and TCR affinity that cantranslate into cross-reactivity and functional avidity of peptide-MHCcomplexes relative to TCR. These parameters can be predictive of the invivo immunologic properties and thus can delineate useful panels ofpeptide analogs to undergo further evaluation, optimization andpractical applications. Analogs that bind to MHC and retaincross-reactivity against TCR specific for the nominal wild-type peptideare predicted to trigger a measurable effect in this assay. The overallmethodology is presented in FIG. 2.

The method used for the generation of T cell lines was the following:HHD transgenic mice carrying an A2 human allele (Pascolo et al., J ExpMed. 185(12):2043-51, 1997, which is hereby incorporated herein byreference in its entirety) were immunized with 50 μg of SSX-2 naturalepitope (41-49) admixed with 25 μg of pIpC at day 0, 4, 14 and 18 bybilateral administration into the inguinal lymph nodes. At 7 days afterthe last boost, the mice were sacrificed and a suspension of splenocytesprepared at 5×10⁶ million cells/ml in complete HL-1 medium. Cells wereincubated with different concentrations of peptide for 48 hours inflat-bottomed 96-well plates (20011/well) and for an additional 24 hourswith rIL-2 at 10 U/ml added to the wells. The supernatant was harvestedand the concentration of IFN-gamma assessed by standard methods such asELISA.

Example 7 Cross-Reactivity and Functional Avidity of Analogs Substitutedat Single Position

The strategy from above (Example 6, FIG. 2) was applied to scan througha library of analogs bearing single substitutions relative to thenatural SSX-2₄₁₋₄₉ epitope (KASEKIFYV (SEQ ID NO. 1)) in its wild-typeversion (FIG. 3). Strong inverse correlation was found between theminimal required amount of analog to elicit IFN-gamma production ex vivoand the maximal amount of cytokine production at any concentration ofanalog.

Substitution of A₄₂ with L, V or M improved on the immunologicproperties of the peptide assessed in this assay. L and V mutants wereactive. M was more active than the natural epitope. The I mutantretained cross-reactivity to the TCR recognizing the wild-type epitope.

Replacement of the A at position 42 with non-standard amino acids Abu,Nle or Nva improved on the immunologic properties of the peptiderelative to the wild-type epitope, both in terms of the minimal amountof analog required to trigger cytokine production and the peak amount ofcytokine produced. Mutants encompassing D-Ala, D-Val, Nal-2 or Aibdisplay retained cross-reactivity and reduced immune activity in thisassay relative to the natural peptide, but can still be useful forfurther derivitization to adjust or enhance their properties. An Nal-1at position 42 abrogated the activity.

Changes of the first residue K₄₁ showed that, while replacement with For Phg improved on the activity, W, D-Lys, and Cha obliterated theimmunologic properties in this assay. Replacement of K with Y, Phe-4F,Phe(4-NO2), O-methyl-Tyr or beta-(3-benzothienyl-Ala) retained activity.

Modification of position V₄₉ (C-terminal residue) by replacement with Iretained the activity at a lower level compared to the original epitope.Modification of the last residue by addition of an NH2 moietyobliterated the activity of the peptide that was subsequently rescued bymodifying the A at position 42 with L or V. This directly illustratesthat analogs with activity that is lower than that of the wild-typepeptide are still useful for further derivatization.

Example 8 Cross-Reactivity and Functional Avidity of Analogs Substitutedat Two Positions

The strategy from Example 6 (FIG. 2) was applied to scan through alibrary of analogs bearing two substitutions, relative to the wild-typeSSX-2₄₁₋₄₉ epitope in its wild-type version (FIG. 4).

Coordinated modifications at position 1 and 2 had a variable effect onthe activity of analogs. For example, substitution of K41 with Y, F or Wcorroborated with substitution of A42 with V, M or I, and resulted inpreserved or enhanced activity of the analogs relative to the wild-typepeptide. Such doubly mutated peptides offer an increased opportunity toimpact the interaction with TCR in a fashion that results in tolerancebreaking (thus being useful for practical application), since the P1residue participates to a certain extent in binding to TCR. Combinationsbetween the following: Y (position 41) with V (at position 42), W(position 41) with I or L (at position 42), and F (position 41) with L,V, I (at position 42) resulted in analogs that were more active relativeto the wild-type peptide. Combinations between Y at position 41 and I atposition 42, or W at position 41 and V or M at 42, conferred an activitysimilar to that of wild-type peptide. Replacement of K with D-lysine atposition 41 reduced resulted in analogs with retained activity in thisassay. Such peptides can be very useful since the metabolic degradationof such peptides encompassing non-standard amino acids is decreased invivo.

Combinations between V or L at position 42 and I at position 49 resultedin increased activity over the natural peptide.

Example 9 Cross-Reactivity and Functional Avidity of Analogs Substitutedat Multiple Positions

The strategy from Example 6 (FIG. 2) was applied to scan through alibrary of analogs bearing three or more substitutions relative to thenatural SSX-2₄₁₋₄₉ epitope in its wild-type version (FIG. 5).

F and V at positions 41 and 42 respectively, combined with I or A atposition 49 resulted in improved or similar activity relative to thewild-type epitope. In contrast, L or M at position 49 resulted inheavily diminished activity.

Triple mutants comprising the non-standard amino acids Nva, Abu or MeValat the last position resulted in retention or improvement of immuneactivity. Such peptides are extremely useful due to increased in vivostability and resistance to enzymatic degradation.

Modification of amino acid residues within the putative TCR bindingregion can result in peptides of considerable value that retain bindingto MHC along with cross-reactivity. Such peptides are useful forrestoration of immune responsiveness or tolerance breaking because theirconformation in the MHC groove is slightly different from that ofnatural peptides. Additional substitutions at position 44 (Q, Nva orNle), position 46 (L, V, Nle or Nva) or 48 (F or Phe-4F) resulted inactive analogs, whereas D, N, S or T at position 44, M at 46 or T, S,Phg at position 48 or L at position 46 with T at 48 resulted in analogsdevoid of activity. Finally, two analogs with 5 substitutions showed noactivity (FIG. 5).

Example 10 Cross-Reactivity and Functional Avidity of DecamersEncompassing the Natural Peptide and Mutated at Various Positions

The strategy from Example 6 (FIG. 2) was applied to scan through alibrary of analogs of a decamer encompassing the nominal SSX-2₄₁₋₄₉peptide (FIG. 6).

The decamer SSX-2₄₁₋₅₀ was significantly less active in stimulating theT cell line specific for the 41-49 nonamer, relative to the latter.Modification of the Y residue at position 50 to I or L, but to a lesseror no extent to V, Nle or Nva, resulted in restoration of activity inthis assay. Further optimization of the activity of decameric analogswas obtained by modification of the A at position 2 with L or V. TheA42L substitution rescued the activity of the Y50Nva decamer. Peptideanalogs of similar or reduced activity in vitro (but retainedcross-reactivity) compared with the natural peptide are still useful forinduction or boost of immune responses due to: i) more limited AICD; ii)potentially higher in vivo activity due to increased stability on classI MHC and/or slightly modified interaction with TCR which can beimportant for tolerance breaking.

Example 11 Use of Analogs to Trigger Enhanced Immunity Against NaturalEpitope, Assessed Ex Vivo

Three groups of mice (n=4) were immunized with a plasmid expressingSSX-2₄₁₋₄₉ natural epitope by direct inoculation into the inguinal lymphnodes with 25 μg in 25 μl of PBS/each lymph node at day 0, 3, 14 and 17.This was followed by two additional peptide boosts (similar amount) atday 28 and 31. The schedule of immunization is shown in FIG. 7. One weekafter the boost, splenocytes were stimulated ex vivo with SSX-2₄₁₋₄₉natural peptide and tested against ⁵¹Cr-labeled target cells (T2 cells)at various E:T ratios (FIG. 8). The results showed that the analog A42Vtriggered a higher response against target cells expressing the naturalpeptide, compared to the analog A42L or the wild-type peptide itself, asboost agents. This correlated with the binding and stability parametersdetermined by ex vivo experimentation.

Example 12 Use of Analogs to Trigger Enhanced Immunity Against NaturalEpitope, Assessed In Vivo

Eight groups of mice (n=4) were immunized with plasmid expressingSSX-2₄₁₋₄₉ natural epitope, by direct inoculation into the inguinallymph nodes with 25 μg in 25 μl of PBS/each lymph node at day 0, 3, 14and 17. This was followed by two additional peptide boosts (similaramount) at day 28 and 31, using a negative control peptide (Melan A26-35 “EAA”), natural peptide or analogs as shown in FIG. 9.

To evaluate the in vivo response against natural peptide, splenocyteswere isolated from littermate control HHD mice and incubated with 20μg/mL or 1 μg/ml of natural peptide for 2 hours. These cells were thenstained with CFSE^(hi) fluorescence (4.0 μM or 11 M for 15 minutes) andintravenously co-injected into immunized mice with an equal number ofcontrol splenocytes stained with CFSE^(lo) fluorescence (0.4 μM).Eighteen hours later the specific elimination of target cells wasmeasured by removing spleen and PBMC from challenged animals andmeasuring CFSE fluorescence by flow cytometry. The relative depletion ofthe populations corresponding to peptide loaded splenocytes wascalculated relative to the control (unloaded) population and expressedas % specific lysis. FIG. 10 (spleen) and 11 (blood) show the in vivocytotoxicity elicited by the regimens described in FIG. 7. Three of thetested peptides, A42V, K41F and K41 Y, showed increased activityrelative to the natural peptide, both in spleen and blood, againsttarget cells coated with 20 μg/ml as well as 1 μg/ml of natural peptide(FIG. 11).

Example 13 Use of Analogs to Trigger Enhanced Responses Against TumorCells

Eight groups of mice (n=4) were immunized with plasmid expressingSSX-2₄₁₋₄₉ natural epitope by direct inoculation into the inguinal lymphnodes with 25 ug in 25 ul of PBS/each lymph node at day 0, 3, 14 and 17.This was followed by two additional peptide boosts (similar amount) atday 28 and 31, using a negative control peptide (Melan A 26-35 “EAA”),natural peptide or analogs as shown in FIG. 9.

One week after the boost, splenocytes were stimulated ex vivo withSSX-2₄₁₋₄₉ wild-type peptide and tested against ⁵¹Cr-labeled human tumorcells (624.38 melanoma cells) at various E:T ratios (FIG. 12). AnalogsA42V and K41F A42V V491 elicited immune responses that mediatedincreased cytotoxicity against human tumor cells expressing the naturalSSX-2₄₁₋₄₉ epitope.

Example 14 N-Terminal Proximal Primary Anchor Modification(2^(nd) AA)

When the substituted analogs shown in Table 3 were tested, the analogsshowed improved binding and stability profiles in comparison with thewild-type peptide epitope. However, the magnitude of improvement foreach analog varied, and the substitution of A42V showed the highestimprovement in terms of binding affinity with HLA-A*0201 molecule.Further, the stability of the A42V-HLA-A*0201 complex was better thanthe complex formed between wild-type peptide and HLA-A*0201: the T1/2extended from 11.5 hrs to 20 hrs. The peptides with 42 A to L, V and Msubstitutions were able to induce the IFN-γ secretion of wild-typepeptide specific CTL at remarkably lower concentrations. The 42A to Isubstitution generated an analog with improved binding and stabilityprofile. The residue at the P2 position can also be engaged in theinteraction with TCR to a certain degree. This observation was alsosupported by the results with the 42 A to Aib analog, which possessed asimilar binding affinity with HLA-A*0201 relative to the wild-typeepitope.

Example 15 N-Terminal Secondary Anchor Modification (1^(st) AA)

The N-terminal secondary anchor is the first amino acid at theN-terminus. Thus, in one embodiment, the original Lys 43 found in thewild-type sequence is substituted with a more hydrophobic and bulkyamino acid. Any more hydrophobic and bulky amino acid also can be used,including any available to or that is known to one of skill in the art,including standard amino acids and non-standard amino acids. Examples ofmore hydrophobic amino acids include, but are not limited to: Phe, Tyr,Trp, and D-Lys.

The residue of Lys 41 was defined as a secondary anchor residue ininteracting with HLA-A*0201 molecule, and it also engaged in theinteraction with the T cell receptors to a certain degree. Therefore,modifications of this position can generate some heteroclitic analogsthat are more immunogenic and more suitable for the development of tumorvaccines.

As shown in Table 3, replacing Lys 41 with Tyr, Phe or Phe derivatives(Phenylglycine, Para-fluorophenylalanine, Para-nitrophenylalanine)resulted in analogs that have higher affinity with the HLA-A*0201molecule and form more stable complexes. On the other hand, the Lys toTrp or Trp derivatives analogs have shown significantly decreasedaffinity with the HLA-A*0201 molecule, although based on the predictedalgorithms, the Trp analog should have a similar affinity to that of theTyr and Phe analogs. The experimental data demonstrate the limitation ofthe predicted algorithms. For examples: Lys 41 to Phg substitutionresulted in an analog with improved affinity and extended stability withthe HLA-A*0201 molecule. The para-nitrophenylalanine analog was shown toinduce the IFN-γ secretion of the wild-type peptide specific CTL at amuch lower concentration, although its affinity with the HLA-A*0201molecule was about the same as that of wild-type peptide.

Example 16 N-Terminal Primary/Secondary Anchor Modification

When both primary and secondary anchor residues at the N-terminal weremodified, a general trend was that resulting analogs demonstratedimproved affinity and extended stability with the HLA-A*0201 molecule(Table 3), with only a few exceptions: (K41 Y, A42V), (K41 Y, A42M) and(K41(D-Lys), A42V). Additionally, they had very good cross-reactivitywith the wild-type peptide specific CTL. Combining the K41 Wsubstitution with A42V or A42L improved the binding/stability profile.These analogs also had desirable cross-reactivity activity with thewild-type peptide. The combination modifications of N-terminal primaryanchor and secondary anchor changed the peptide structure andconformation to a greater degree.

Example 17 N-Terminal Primary/Secondary Anchor and C-Terminal PrimaryModification

The C-terminal Val of the wild-type peptide was a preferred anchorresidue. However, improved potency was observed when it was mutated toIle, having one additional —CH2 group. Similar improvement was alsoobserved with a Val to Abu substitution. The other analogs showedimproved binding affinity and stability with the MHC molecule. Theresults of these analogs indicated that the peptide C-terminal anchorresidue also plays a critical role in the recognition of T cells. (Table4).

Example 18 N-Terminal Primary/Secondary Anchor and Tcr SitesModification

Substitutions of secondary TCR binding amino acid residues generatedheteroclitic analogs that did not interfere with the binding to the MHCmolecule, but overcame the tolerance issues of self-antigens. Bycombining the substitutions of N-terminal primary/secondary anchorresidues (K41 F and A42V) and the TCR sites, analogs were generated withimproved binding affinity and stability (Table 6). Some of these analogsinduced the IFN-γ production of the wild-type peptide specific CTL atlower concentrations, such as K41F, A42V, E44(Nva)/(Nle) mutants andK41F, A42V, 146L/(Nva)/(Nle) mutants.

Example 19 N-Terminal Amide

Replacing the peptide's free carboxylic acid C-terminus with an amideimproved the peptide's stability in biological media by conferringstability to proteolysis and conferred dipeptidyl carboxypeptidaseresistance to the peptide. However, some of the resultant analogs lost asignificant amount of their affinity with MHC molecules, as well asimmunogenicity and antigenicity. Unexpectedly, although the threeanalogs disclosed herein at Table 7 lost their binding capability withMHC molecule, SSX-2₄₁₋₄₉-NH2 (A42V) retained its reactivity withwild-type peptide specific CTLs as indicated by its capability ofinducing the secretion of IFN-γ at a similar concentration to that ofthe wild-type peptide. SSX-2₄₁₋₄₉ —NH2 (A42L) was, however, able tostimulate the IFN-γ production at a lower concentration.

Example 20 Decamers

The length of typical MHC binding peptides varies from 8-mer to 11-mer,and most HLA-A*0201 binding peptides are 9-mers or 10-mers. In previousobservations, a 9-mer and 10-mer from a natural sequence were both foundto possess a binding motif for the same MHC, and had the sameN-terminus. From the standpoint of proteasomal processing they aredistinct epitopes, but were nonetheless antigenically cross-reactive. Inthe case of the wild-type epitope SSX-2₄₁₋₄₉, the epitope is a 9-merpeptide and the 10-mer peptide, SSX-2₄₁₋₅₀, lacks the appropriate MHCbinding motif and showed no immunological activity. The wild-typeepitope was therefore lengthened to a 10-mer with amino acids that couldcreate the appropriate binding motif. As shown in Table 8, while many10-mer analogs have a lower binding affinity with the HLA-A*0201molecule, analogs SSX-2₄₁₋₅₀ (A42L, Y50L/V/Nle/Nva) showed improvedbinding affinity with the HLA-A*0201 molecule. Two 10-mer analogs inparticular, A42L and Y50 Nle/Nva, were able to induce IFN-γ productionfrom T cells immunized against the wild-type peptide at lowerconcentrations than the wild-type peptide.

Example 21 Use of Analogs to Overcome Tolerization

One aspect in which the analogs can represent an improvement over thewild-type epitope is in increased immunogenicity in a human system andtolerance breaking. Differences in the TCR repertoire, whether due togerm line differences or differences in negative selection, have thepotential to give anomalous results. To address such issues, the analogswere used in an in vitro immunization of HLA-A2⁺ blood to generate CTL.Techniques for in vitro immunization, even using naive donors, are knownin the field, for example, Stauss et al., Proc. Natl. Acad. Sci. USA89:7871-7875, 1992; Salgaller et al., Cancer Res. 55:4972-4979, 1995;Tsai et al., J. Immunol. 158:1796-1802, 1997; and Chung et al., J.Immunother. 22:279-287, 1999; each of which is hereby incorporated byreference in their entirety.

Specifically, PBMCs from normal donors were purified by centrifugationin Ficoll-Hypaque from buffy coats. All cultures were carried out usingautologous plasma (AP) to avoid exposure to potential xenogeneicpathogens and recognition of FBS peptides. To favor the in vitrogeneration of peptide-specific CTL, autologous dendritic cells (DCs)were employed as APCs. DCs were generated and the CTLs were induced withDCs and peptides from PBMCs as described in Keogh et al., 2001, which isincorporated herein by reference in its entirety. Briefly,monocyte-enriched cell fractions were cultured for 5 days with GM-CSFand IL-4 and were cultured for 2 additional days in culture media with 2μg/ml CD40 ligand to induce maturation. 2×10⁶ CD8+-enriched Tlymphocytes/well and 2×10⁵ peptide-pulsed DCs/well were co-cultured in24-well plates in 2 ml RPMI supplemented with 10% AP, 10 ng/ml IL-7 and20 IU/ml IL-2. Cultures were restimulated on days 7 and 14 withautologous irradiated peptide-pulsed DCs. Immunogenicity was assayedusing the in vitro cytotoxicity and cytokine production assays describedherein.

Examples 22-30 Testing of Ny-Eso-1₁₅₇₋₁₆₅ Analogs

The analogs listed in FIG. 13 were tested for activity, such as bindingand biological effect as follows in Examples 22-30:

Example 22 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at a Single Position (FIGS. 13 A-C).

The strategy from above (Example 6) was applied to scan through alibrary of analogs bearing single substitutions relative to thewild-type NY-ESO-1₁₅₇₋₁₆₅ epitope in its native (or wild-type) version(FIG. 13). A strong inverse correlation was found between the minimalrequired amount of analog to elicit IFN-gamma production ex vivo and themaximal amount of cytokine production at any concentration of analog.

Substitution of S157 with F or K resulted in analogs that partiallyretained MHC binding and cross-reactivity with the T cells specific forthe nominal epitope. Substitution of LI 58 with I improved theimmunologic features of the peptide as assessed by this methodology;whereas L158V resulted in partial retention of activity. Modification ofC165 with any of the amino acids V, L, A, or I resulted in improvedimmune properties.

Peptides that have substitutions in the N-terminal position orelsewhere, and present with retained but not increased activity in thisassay relative to the wild-type peptide, can be useful in humans. Inaddition, they are material for further derivatization to improve ontheir properties, as described below.

Example 23 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Two Positions (FIG. 13A-C).

The strategy from Example 6 was applied to scan through a library ofanalogs bearing two substitutions relative to the wild-typeNY-ESO-1₁₅₇₋₁₆₅ epitope. Simultaneous semi-conservative modifications atposition 2 and 9 were found to have profound effects on the immuneproperties of analogs, depending on the precise identity of the analogs.Combining L158I with C165V or C165L further increased activity relativeto the wild-type peptide. Similarly, L158V improved on the activity ofthe C165V or C165L analogs, further increasing such activity relative towild-type peptide. L158V partially retained the activity of C165A orC165I analogs, showing an interesting effect of double mutation ofprimary anchor residues. Similarly, L158I partially retained theactivity of the C165A analog.

Simultaneous modifications at positions 1 and 9 had profound effects onthe immune properties of analogs, depending on the precise identity ofthe analogs. S157Y combined with C165Nva (norvaline) or Nle (norleucine)at position 9 resulted in substantially improved activity over S157Yalone or the wild-type peptide. The C165V mutant also rescued theactivity of the S157Y mutant. V—NH2 or L-NH2 at position 9 partiallyrescued the activity of the S157Y analog, however, A-NH2 failed to doso. Combinations between S157F and V, L, I, and to a lesser extent A atthe 9^(th) position retained strong activity of the analog and may bemore useful than single mutants at position 9 due to the participationof the first residue in the interaction with TCR. Combinations betweenS157K and V, L, I and to a lesser extent A at the 9^(th) position,retained strong activity of the analog and may be more useful thansingle mutants at position 9 due to the participation of the firstresidue in the interaction with TCR and the overall beneficial effect onthe peptide solubility of K at position 1.

Example 24 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at multiple positions (FIG. 13A-C).

The strategy from Example 6 was applied to scan through a library ofanalogs bearing multiple substitutions relative to the wild-typeNY-ESO-1 epitope.

L158Nva or L158Nle considerably improved on the activity of the S157YC165V mutant. Combinations between V or I at position 158 and V, L, A orI at 165 partially restored the potency of analogs relative to thewild-type peptide. S157Y L158I C165V displayed increased activityrelative to the wild-type peptide and S157V with C165V or C165I; andS157I with C165L or I retained MHC binding and cross-reactivity with Tcells specific for the wild-type peptide.

Triple substitutions comprising Y and V at positions 157 and 165,respectively, in addition to L or N at 160; A, L, V, or N at 162; or E,D or T at 164, retained the activity of the peptide in thiscross-reactivity assay, making these analogs useful compounds forbreaking T cell tolerance in vivo as positions 160, 162 and 164participate in the interaction with TCR.

Triple substitutions comprising 157F and 158V plus V, L, or I at theposition 165 showed activity in the assay described in Example 2. Inaddition, triple mutants encompassing S157F and L158I plus V or A atposition 165 retained activity. Together, these data underline thecomplex interactive and non-linear nature of multiple substitutions.

Finally, triple mutants comprising S157W and to a higher extent S157Ttogether with 158V and 165V, showed retained or increased activity,respectively, relative to the wild-type peptide.

Example 25 Cross-Reactivity and Functional Avidity of DecamersEncompassing the Wild-Type Peptide and Mutated at Various Positions(FIG. 13A-C).

The strategy from Example 6 was applied to scan through a library ofanalogs of a decamer encompassing the nominal NY-ESO-1₁₅₇₋₁₆₅ peptide.While the decamer itself lacked significant in vitro activity, varioussubstitutions at this position partially rescued activity, such as L at166, or L, I, Nle at 166 combined with Y at 157 and Vat 165.

Peptide analogs with similar or reduced activity in vitro (but withretained cross-reactivity) compared to the wild-type peptide are stilluseful for induction or boost of immune responses due to: i) morelimited AICD (antigen-induced cell death); ii) higher in vivo activitydue to increased stability on class I MHC and/or slightly modifiedinteraction with TCR. Thus, these analogs are useful for breakingtolerance.

Example 26 Evaluation of Immunologic Properties of Analogs: PeptideBinding to MHC Class I Molecules (FIG. 13A-C)

The affinity of peptide analogs and the wild-type epitope to HLA-A*0201was evaluated by T2 cell based assay (Regner M, et al., Exp ClinImmunogenet. 1996; 13(1):30-5, which is incorporated herein by referencein its entirety). For the binding assay, in brief, T2 cells that lackexpression of TAP, and thus do not assemble stable MHC class I on thecell surface, were pulsed with different concentrations of peptides(controls or analogs) overnight at 37° C., washed extensively, stainedwith fluorescently tagged antibody recognizing MHC class I (A2 allele),and run through a FacsScan analyzer. Peptides that bind A2 stabilize itspresence at the cell surface. The difference between the MFI (meanfluorescence intensity) corresponding to a given concentration of analogand the negative control (a non-MHC binding peptide) is a function ofhow many stabilized complexes between MHC and peptide are displayed onthe surface of T2 cells. Thus, at limiting concentrations of peptide,this is a measurement of K_(on) mostly and at saturation levels ofpeptide that is a measurement of both K_(on) and K_(off).

In FIG. 13, the binding is quantified by two factors that aremathematically related: Half maximal binding (the peptide concentrationgiving 50% of the signal corresponding to saturation) and relativeaffinity (1/RA), that is binding normalized to a reference (wild-typepeptide)—i.e., the ratio between half maximal binding of controlrelative to peptide analog. The higher the 1/RA index and the lower thehalf maximal binding, the higher the K_(on) of the interaction betweenan analog and the MHC molecules. In FIG. 13, there are 39 analogsdescribed with such binding parameters improved relative to thewild-type peptide. Such improved binders carry one, two, three, or moresubstitutions of standard and/or non-standard amino acids at positionsthat are known to participate in the interaction with MHC and/or TCR,with an overall effect on MHC binding that is dependent onprecise/conjugated modification. Such peptide analogs are useful intherapeutic compositions or as a platform to further derive therapeuticcompositions.

Example 27 Method of Immunization (FIG. 14)

Eight groups of mice (n=4) were immunized with a plasmid expressing thewild-type NY-ESO-1₁₅₇₋₁₆₅ epitope by direct inoculation into theinguinal lymph nodes with 25 μg in 25 μl of PBS into each lymph node atdays 0, 3, 14 and 17. This was followed by two peptide boosts (similaramount) at day 28 and 31, using a negative control peptide (HBVc),wild-type peptide or analog as shown in FIG. 14.

Example 28 Use of Analogs to Trigger Enhanced Immunity Against Wild-TypeEpitope, Assessed In Vivo (FIGS. 15A-C)

To evaluate the in vivo responses obtained against the wild-typeepitope, splenocytes were isolated from littermate control HHD mice andincubated with 20 μg/mL or 1 μg/ml of wild-type peptide for 2 hours.These cells were then stained with CFSE^(hi) and CFSE^(med) fluorescence(4.0 μM or 1 μM, respectively, for 15 minutes) and intravenouslyco-injected into immunized mice with an equal number of controlsplenocytes stained with CFSE^(lo) fluorescence (0.4 μM). Eighteen hourslater the specific elimination of target cells was measured by removingthe spleens and PBMC from challenged animals and measuring CFSEfluorescence by flow cytometry. The relative depletion of thepopulations corresponding to peptide loaded splenocytes was calculatedrelative to the control (unloaded) population and expressed as %specific lysis. FIG. 15A shows the lack of in vivo cytotoxicity in micereceiving the negative control peptide. FIG. 15B shows the variablecytotoxicity in mice immunized with plasmid and amplified with wild-typepeptide. FIG. 15C shows the substantial, constant cytotoxicity in miceimmunized with plasmid and amplified with the analog L158Nva C165V.

Example 29 Comparison of Various Analogs in Triggering Enhanced ImmunityAgainst the Wild-Type Epitope, Assessed In Vivo (FIGS. 16A-B)

In the context of the immunization protocol described in Example 8 andusing the methodology described in the Example 9, in vivo cytotoxicityagainst target cells coated with limited (1 μM; FIG. 16A) orsupraoptimal amounts of wild-type peptide (20 μM, FIG. 16B) wasevaluated subsequent to the entrain and amplify protocol using plasmidand peptide analog respectively for the two stages. Results expressed asaverage % specific lysis +/− SE showed that the analog L158V C165Nvainduced the highest activity and that the analogs L158V C165V, L158VC165Nva and S157K L158V C165V showed an effect in the same range withwild-type peptide or the C165V mutant. Because multiple substitutionscan alter the TCR binding site, such analogs can be more useful than thewild-type peptide in breaking tolerance against a self epitope. Inaddition, the S157K triple mutant can ameliorate the poor solubility ofthe wild-type peptide or other analogs with direct practicalimplications.

Example 30 Use of Analogs to Trigger Enhanced Immunity Against theWild-Type Epitope, Assessed Ex Vivo by Cytokine Production (FIGS. 17A-B)

In the context of the immunization protocol described in Example 27, andfollowing the challenge described in Example 28, splenocytes wereisolated, pooled and stimulated in vitro with 10 μM of wild-type peptideNY-ESO-1₁₅₇₋₁₆₅ for 3 and 6 days, respectively. Supernatants wereharvested and the concentration of IFN-γ measured by ELISA.

Analog L158Nva C165V induced T cells that produced large levels ofIFN-gamma more rapidly upon ex vivo stimulation (FIG. 17A). Otheranalogs such as S157F L158V C165V, L158V C165Nva, and L158V C165Vinduced T cells that produced increased amounts of IFN-gamma upon exvivo re-stimulation with wild-type peptide (FIG. 17B). In contrast,C165V failed to induce increased capability of T cells to produce IFN-γ,relative to the wild-type peptide following the protocols described inExamples 27-28.

Example 31 Characterization of Binding and Stability by ELISA (ItopiaTesting)

Avidin-coated microtiter plates containing class I monomer loaded with aso-called placeholder peptide were used to evaluate peptide binding,affinity and off-rate. The monomer-coated plates were supplied as partof the iTopia Epitope Discovery System Kit (Beckman Coulter, Inc., SanDiego, Calif., USA). Assay buffers, anti-MHC-FITC mAb andbeta2-microglobulin and control peptides were also supplied with thekits.

Binding Assay:

Native peptide and analogs were first evaluated for their ability tobind each MHC molecule by binding assay. This assay measures the abilityof individual peptides to bind HLA molecules under standardized optimalbinding conditions. Monomer-coated plates were first stripped, releasingthe placeholder peptide and leaving only the MHC heavy chain bound tothe plate. Test peptides were then introduced under optimal foldingconditions, along with the anti-MHC-FITC mAb. Plates were incubated for18 hours at 21° C. The anti-MHC-FITC mAb binds preferentially to arefolded MHC complex. Therefore, the fluorescence intensity resultingfrom each peptide was related to the peptide's ability to form complexwith MHC molecule. Each peptide's binding was evaluated relative to thepositive control peptide provided in the kit, and the results wereexpressed “% binding”. The analogs identified as ‘better-binders’relative to the native peptide were subsequently analyzed in theaffinity and off-rate assays.

Affinity Assay:

For the affinity assay, after the initial stripping of the placeholderpeptide, increasing concentrations (range 10⁻⁴ to 10⁻⁸ M) of each testpeptides for a given allele were added to a series of wells andincubated under the conditions described previously. Plates were read onthe fluorometer. Sigmoidal dose response curves were generated usingPrism software. The amount of peptide required to achieve 50% of themaximum was recorded as ED₅₀ value.

Off-Rate Assay:

For the off-rate assay, the plates were washed after 18 hrs incubationat 21° C. to remove excess peptide. The plates were then incubated onthe allele-specific monomer plates at 37° C. The plates were measured atmultiple time points (0, 0.5, 1, 1.5, 2, 4, 6 and 8 hrs) for relativefluorescence intensity. The time required for 50% of the peptide todissociate from the MHC monomer is defined as the T1/2 value (hrs).

iScore Calculation:

The iScore is a multi-parameter calculation provided within the iTopiasoftware. Its value was calculated based on the binding, affinity andstability data.

Example 32 Validation of the Antigenicity of PSMA₂₈₈₋₂₉₇

HHD transgenic mice (n=4) were immunized with PSMA₂₈₈₋₂₉₇ peptide (25 μgin 25 μl of PBS, plus 12.5 μg of pI:C to each lymph node) at day 0, 3,14 and 17. One week after the boost, splenocytes were stimulated ex vivowith the native PSMA₂₈₈₋₂₉₇ peptide and tested against ⁵¹Cr-labeledhuman tumor cells (PSMA⁺ A2⁺ LnCap cells, or as negative control, LnCapcells coated with MHC class I-blocking antibody) at various E:T ratios.The results, expressed as % specific lysis (mean±SEM), showed thatPSMA-specific T cells were able to lyse human tumor cells in a fashiondependent on MHC class I availability, confirming display of the PSMAepitope on MHC class I of tumor cells in a fashion allowing immunemediated attack (FIG. 18).

Examples 33-38 Testing of PSMA₂₈₈₋₂₉₇ Analogs

The analogs listed in FIGS. 19 and 20 were tested for various propertiessuch as improved affinity and stability of binding, cross-reactivitywith the native epitope, and immunogenicity as follows in Examples33-38.

Example 33 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Single Position

Using the procedures described in Example 31, the bindingcharacteristics of PSMA₂₈₈₋₂₉₇ and analogs were assessed in comparisonto each other (see FIG. 19). The positive control for binding wasmelan-A₂₆₋₃₅ A27L. Cross reactivity with the native epitope was assessedby using the analog peptides to stimulate IFN-gamma secretion from a Tcell line specific for the native epitope, essentially as described inExample 6. The data shown in FIG. 19 was generated by stimulating with10 μg/ml of analog (approximately 10 μM). This concentration generallyresulted in maximal or near-maximal IFN-gamma production for the analogsand thus was chosen to represent cross-reactivity.

The observed affinities of the analogs are reported in FIG. 19 as ED₅₀s.Met, Ile, Gln, Val, Nva, Nle, and Abu were substituted at the P2position, and generally resulted in similar affinity. The Nle and Metsubstitutions also maintained similar stability of binding, measured ashalf-time of dissociation in hours. The Val, Nva, and Abu analogselicited a similar level of IFN-gamma production.

Val, Leu, Nva, and Nle were substituted for the Ile at the PΩ primaryanchor position. All four had similar binding affinity. The Val and Nvasubstitutions improved the stability of binding and increased the amountof IFN-gamma produced, indicating cross-reactivity and that the analogscan have improved immunogenicity.

The Ser, Ala, Sar, and Abu substitutions at P1 maintained similarbinding characteristics but had marginally similar cross-reactivity. TheAla, Leu, Ser, and Thr substitutions at the PΩ-1 position alsomaintained similar binding characteristics. Finally, the Trpsubstitution at P3 exhibited affinity and stability of binding that wereboth increased about twofold and IFN-gamma production that was withintwofold of the native peptide, all generally similar values.

Example 34 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Two Positions

The pattern seen above, that substitutions in this epitope did notgreatly impair binding affinity, continued with the double substitutionsexamined (FIG. 20) which uniformly displayed similar or improved bindingaffinity compared to the native peptide. Among the analogs withsubstitutions at both primary anchor positions, those with Nva of Nle atP2 and Val at PΩ, and Val at P2 and Nva at PΩ displayed improved bindingstability and the former two increased IFN-gamma production (data notshown for the 3^(rd) analog). The Val and Nva substitutions at PΩ werealso paired with Ala and Abu substitutions at P1. These analogs all hadrobust binding stability and IFN-gamma production that was improvedcompared to the single PΩ substitutions, thus further improving the P1substitutions. The PΩ Nva substitution was also able to maintain bettercross-reactivity than PΩ V when combined with the P3 Trp substitution,although the various binding parameters were generally similar.

Example 35 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Three Positions

Triple substitutions at P1, P2, and P3; P1, P2, and PQ; P2, P3, and PΩ;and P1, P3, and PΩ were made (FIG. 21). In all cases, the P1substitution was Ala, the P3 substitution was Trp, and the PΩsubstitution Val or Nva. As above, affinity at least similar to thenative peptide was maintained. For the P1, P2, P3 class Nva and Nle atP2 improved the stability of binding. This P2 Nva analog elicited asimilar amount of IFN-gamma while the Nle analog showed a substantialincrease.

For the P1, P2, PΩ class, Nva and Val at P2 and PΩ in either combinationimproved binding stability. This P2 Nva PΩ Val analog also showed asubstantial increase in IFN-gamma production. (Data not shown). Val atboth P2 and PQ in this triple substitution showed binding stability andIFN-gamma production that was nearly halved from that of the nativepeptide.

For the P2, P3, PΩ group, only the Nva/W/V analog showed improvedbinding or IFN-gamma production. For the two P1, P3, PΩ analogs examinedPΩ of Val or Nva improved binding stability.

Example 36 Cross-Reactive Immunogenicity of Various Analogs

Groups of HHD transgenic mice (n=8) were immunized with peptide (naturalepitope PSMA₂₈₈₋₂₉₇, or analogs bearing substitutions at primary orsecondary anchor residues) by direct inoculation into the inguinal lymphnodes, with 25 μg in 25 μl of PBS+12.5 μg of pI:C to each lymph node atday 0, 3, 14 and 17.

Mice were sacrificed at 10 days after the last boost, and splenocytesprepared and assessed for IFN-γ production by ELISPOT analysis. Variousnumbers of splenocytes/well were stimulated with 10 μg/ml of nativepeptide in ELISPOT plates coated with anti-IFN-γ antibody. At 48 hoursafter incubation, the assay was developed and the frequency ofcytokine-producing T cells that recognized native PSMA₂₈₈₋₂₉₇ peptidewas automatically counted. The data is represented in FIG. 22 as thenumber of spot forming colonies/well (mean of triplicates±SD). The datashow increased priming of immune responses against the native epitopeachieved by the I297V and P290W analogs, with the other analogs showingslightly higher (but significant) activity than the native peptide(I297Nva or G288Abu or L289Nle I297Nva). To the extent that the poorimmunogenicity of the native epitope reflects tolerance, the improvedactivity of these analogs represents tolerance breaking.

Example 37 Amplification by the I297V Analog of the Response toPSMA₂₈₈₋₂₉₇ Induced by Plasmid

Two groups of HHD transgenic mice (n=8) were immunized with plasmidexpressing PSMA₂₈₈₋₂₉₇ by direct inoculation into the inguinal lymphnodes with 25 μg in 2511 of PBS to each lymph node at day 0, 3, 14 and17. This was followed by two peptide boosts (25 μg) at day 28 and 31with either the natural peptide or the I297V analog.

Mice were sacrificed at 10 days after the last boost, and splenocytesprepared and assessed for IFN-γ production by ELISPOT analysis. Variousnumbers of splenocytes/well were stimulated with 10 μg/ml of nativepeptide in ELISPOT plates coated with anti-IFN-γ antibody. At 48 hoursafter incubation, the assay was developed and the frequency ofcytokine-producing T cells that recognized the PSMA₂₈₈₋₂₉₇ peptide wasautomatically counted. The data is represented in FIG. 23 as frequencyof specific T cells normalized to 0.5 million responder cells (mean oftriplicates+SD). The data show that irrespective of the number ofsplenocytes/well, the frequency of native epitope-specific T cells wasconsiderably higher in the mouse group immunized with the I297V analog.

Example 38 Ex Vivo Cytotoxicity Against Human Tumor Cells

HHD transgenic mice (n=4) were immunized with plasmid expressing thePSMA₂₈₈₋₂₉₇ epitope by direct inoculation into the inguinal lymph nodeswith 25 μg in 25 μl of PBS to each lymph node at day 0, 3, 14 and 17.This was followed by two peptide boosts (same amount) at day 28 and 31,with the analog I297V. One week after the boost, splenocytes werestimulated ex vivo with the native PSMA₂₈₈₋₂₉₇ peptide and testedovernight against ⁵¹Cr-labeled human tumor cells (Lncap, A2⁺ PSMA⁺; or624.38 A2⁺ PSMA⁻ or control 624.28 cells A2⁻ PSMA⁻) at various E:Tratios. The resulting immunity was effective in mediating cytotoxicityagainst LNCAP (FIG. 24).

Example 39 Validation of the Antigenicity of PRAME₄₂₅₋₄₃₃

HHD transgenic mice (n=4) were immunized with PRAME₄₂₅₋₄₃₃ peptide (25μg in 25 μl of PBS, plus 12.5 μg of pI:C to each lymph node) at day 0,3, 14 and 17. One week after the boost, splenocytes were stimulated exvivo with the native PRAME₄₂₅₋₄₃₃ peptide and tested against⁵¹Cr-labeled human tumor cells (PRAME⁺ A2⁺ 624.38 melanoma cells; ornegative control 624.38 cells, deficient in A2 expression) at variousE:T ratios. The results, expressed as % specific lysis (mean±SEM),showed that PRAME-specific T cells were able to lyse human tumor cells,confirming display of the PRAME₄₂₅₋₄₃₃ epitope on MHC class I of tumorcells in a fashion allowing immune mediated attack (FIG. 25).

Examples 40-48 Testing of PRAME₄₂₅₋₄₃₃ Analogs

The analogs listed in FIGS. 26-28 were tested for various propertiessuch as improved affinity and stability of binding, cross-reactivitywith the native epitope, and immunogenicity as follows in Examples40-48. Using the procedures described in Example 31, the HLA-A*0201binding characteristics of PRAME₄₂₅₋₄₃₃ and 69 analogs were assessed incomparison to each other. The positive control for binding wasmelan-A₂₆₋₃₅ A27L. The observed affinities of the analogs are reportedas % binding (compared to the positive control) and ED₅₀, and stabilityof binding as half time of dissociation. Cross reactivity with thenative epitope was assessed by using the analog peptides to stimulateIFN-gamma secretion from a T cell line specific for the native epitope,essentially as described in Example 6. The data shown in FIGS. 26-28were generated by stimulating with analog peptide at approximately 0.3μM. The results were collected from three separate experiments and werenormalized to the amount of IFN-γ elicited by the native peptide ineach. In some cases, the reported values are the average of twodeterminations. An asterisk “*” indicates that IFN-γ production was notdistinguishable from background.

Example 40 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at a Single Position (FIG. 26)

Single substitutions of Val, Met, Ile, Nle, Nva, and Abu were made forthe Leu at the P2 primary anchor position. All of these analogsexhibited % binding within 20% of the native peptide. The ED50 for theMet analog had an affinity comparable to the native peptide while theNva and Ile analogs' affinities were reduced within about 3-fold, butwere still comparable to the PSMA₂₈₈₋₂₈₇ epitope. All of the P2substitutions maintained binding stability at least similar to thenative peptide. The Met, Nle, and Nva analogs elicited IFN-γ productionwithin twofold of the native peptide and the Val analog somewhat less.

Single substitution of Lys, Phe, Tyr, Thr, Orn (ornithine), and Hse(homoserine) were made for the Ser at the P1 position. All of theseanalogs exhibited % binding within 20% of the native peptide except forthe Phe analog which exceeded that range on the high side. The ED50 forthe Lys analog was reduced by almost 6-fold, but the other five analogshad affinities within threefold of the native peptide. Stability ofbinding was generally similar to the native peptide with the Phe P1analog showing greatest binding stability in this group with a half timeof dissociation of 17.7 hours compared to 12.2 hours for the nativepeptide. With the exception of the Lys P1 analog, which elicited 40% ofthe IFN-γ of the native peptide, all of these analogs were consideredcross-reactive as they elicited IFN-γ production within twofold of thenative peptide.

Single substitutions of Val, Ile, Ala, Nle, Nva, Abu, were made to thePΩ anchor position, as well as modifying the carboxy-terminus by theaddition of an amide group. All of these analogs exhibited % bindingwithin 20% of the native peptide. ED50 measurements ranged from morethan 10-fold less for the Ala substitution to a comparable value for theNle substitution; the Nva substitution and C-terminal amide were alsowithin 3-fold of the ED50 for native peptide. Stability of binding wasalso generally similar with outliers of the Nva analog at the high end,t1/2 of 17.2 hours, and the C-terminal amide at the low end with asignificantly reduced t1/2 of only 3 hours. The Val, Ile, Ala, and AbuPΩ analogs exhibited less preferred cross-reactivity, but the otherselicited IFN-γ production within twofold of the native peptide.

Single substitutions at positions primarily affecting TCR interactionswere also made: Nle, Nva, and Abu at P3 and P6, and Ala, Ser, and Sar atP8. The P6 Nva analog produced IFN-γ within twofold of that of thenative peptide, though the P6 Abu analog was close at 44%.

Example 41 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Two Positions

Double substitution analogs were created at P1 and P2, P2 and PΩ, and P1and PΩ using various combinations of the single substitutions above(FIGS. 27A and 27B). None of the P1-P2 double substitutions examined hadradical changes to binding affinity or stability, but none exhibitedsignificant cross-reactivity in the IFN-γ assay. A similar pattern wasseen with the P2-PΩ double substitution analogs, however, the L426NvaL433Nle analog exhibited a significant level of cross-recativity withthe native peptide in the IFN-γ assay along with its similar, somewhatimproved binding characteristics. Finally, for the P1-PΩ doublesubstitutions, the examined analogs also conformed to the generalpattern of having at least similar binding characteristics, buteliciting negligible IFN-γ in the cross-reactivity assay. The exceptionsin this grouping were the S425F L433Nle analog, which exhibited somewhatimproved binding stability and significant cross-reactivity, and theS425F L433Nle analog, which had a more that fourfold reduced ED₅₀, anearly doubled halftime of dissociation, and elicited more IFN-γ thanthe native peptide.

Example 42 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Three Positions

Four triple substitution analogs were investigated, having Phe or Thr atP1, Nva or Met at P2, and Nle at PΩ. The S425T L426M L433Nle analog hadsimilar affinity whereas the affinity was improved for the other threeanalogs. Both analogs with P2 Nva substitutions displayed increasedstability of binding and significant levels of cross-reactivity. SeeFIG. 28.

Example 43 Cross-Reactive Immunogenicity of the L426Nva L433Nle Analog

Two groups of HHD transgenic mice (n=8) were immunized with a pCTLR2plasmid expressing PRAME₄₂₅₋₄₃₃, described in example 49 below, bydirect inoculation into the inguinal lymph nodes of 25 μg in 25 μl ofPBS to each lymph node at day 0, 3, 14 and 17. This was followed by twopeptide boosts (2.5 μg) at day 28 and 31, of native peptide or the PRAMEepitope analog L426Nva L433Nle.

Mice were sacrificed at 10 days after the last boost, and splenocytesprepared and assessed for IFN-γ production after in vitro stimulation at0.5×10⁶ cells/well, with 10 μg/ml of native peptide. At 48 hours afterincubation, the supernatant was harvested and the concentration of IFN-γproduced in response to the PRAME₄₂₅₋₄₃₃ peptide was measured by ELISA.The data are presented in FIG. 29 and show a significant enhancement ofIFN-γ production in mice boosted with the PRAME₄₂₅₋₄₃₃ L426Nva L433Nleanalog.

Example 44 In Vivo Cytotoxicity Induced by the L426Nva L433Nle Analog

Two groups of HHD transgenic mice (n=8) were immunized as described inExample 43 above.

To evaluate the in vivo responses obtained against the native epitope,splenocytes were isolated from littermate control HHD mice and incubatedwith 20 μg/mL or 1 μg/ml of native peptide for 2 hours. Cells were thenstained with CFSE^(hi) and CFSE^(med) fluorescence (4.0 μM or 1 μM,respectively, for 15 minutes) and intravenously co-injected intoimmunized mice with an equal number of control splenocytes stained withCFSE^(lo) fluorescence (0.4 μM). Eighteen hours later the specificelimination of target cells was measured by removing the spleens andPBMC from challenged animals and measuring CFSE fluorescence by flowcytometry. The relative depletion of the populations corresponding topeptide loaded splenocytes was calculated relative to the control(unloaded) population and expressed as % specific lysis. The results inFIG. 30 showed preserved induction of cytotoxicity when the analogreplaced the natural peptide as a booster agent. The trend indicatesthat the analog can improve on induction of cytotoxic immunity.

Example 45 In Vivo Cytotoxicity and Tetramer Staining

Seven groups of HHD transgenic mice (n=4) were immunized with a plasmid,pCTLR2, expressing PRAME₄₂₅₋₄₃₃ by direct inoculation into the inguinallymph nodes of 25 μg in 25 μl of PBS to each lymph node at day 0, 3, 14and 17. This was followed by two peptide boosts (2.5 μg) at day 28 and31, of native peptide, negative control (EAAGIGILTV peptide (SEQ ID NO.100)), or PRAME₄₂₅₋₄₃₃ epitope analogs bearing mutations at the primaryand/or secondary anchor residues —S425F, L426Nva L433Nle, S425T L433Nle,and S425T L426Nva L433Nle.

To evaluate the in vivo response against native peptide, splenocyteswere isolated from littermate control HHD mice and incubated with 0.2μg/ml or 20 μg/ml of native peptide for 2 hours. These cells were thenstained with CFSE fluorescence (1 and 2.5 μM, respectively, for 15minutes) and intravenously co-injected into immunized mice with an equalnumber of control splenocytes stained with CFSE^(lo) fluorescence (0.4μM). Eighteen hours later, the specific elimination of target cells wasmeasured by removing the spleen from challenged animals and measuringCFSE fluorescence in the resulting cell suspensions, by flow cytometry.The relative depletion of the populations corresponding topeptide-loaded splenocytes was calculated relative to the control(unloaded) population and expressed as % specific lysis. In addition,the frequency of PRAME₄₂₅₋₄₃₃-specific T cells was evaluated bytetramer/CD8 co-staining.

The boost with analogs encompassing mutations at primary or secondaryanchor residues showed comparable immune activity as compared to thenative peptide, based on in vivo cytotoxicity and tetramer staining. Theanalogs were capable of amplifying the immune response, as shown bycomparison with the “EAA” group, boosted with an irrelevant peptide. Inthat regard, analogs comprising S425F, L33Nle, L426Nva L433Nle, S425TL433Nle, or S425T L426Nva L433Nle were all capable of expanding theimmunity against the native epitope, as assessed by in vivocytotoxicity. However, only the L433Nle, L426Nva L433Nle, and S425TL426Nva L433Nle analogs expanded the subset of T cells specific againstthe native epitope to a level significantly higher than in mice primedwith plasmid and boosted with the negative control peptide (FIG. 31).

Example 46 Ex Vivo Cytokine Production

Three groups of HHD transgenic mice (n=4) were immunized with a plasmid,pCTLR2, expressing PRAME₄₂₅₋₄₃₃ by direct inoculation into the inguinallymph nodes of 25 μg in 25 μl of PBS to each lymph node at day 0, 3, 14and 17. This was followed by two peptide boosts (2.5 μg) at day 28 and31, of the PRAME epitope analogs L426Nva L433Nle and S425T L426NvaL433Nle or the negative control peptide Melan A (EAAGIGILTV (SEQ ID NO.100)).

Mice were sacrificed at 10 days after the last boost, and splenocytesprepared and assessed for IFN-γ production by ELISA at 48 hours afterincubation with 10 μg/ml of native peptide. The data are presented inFIG. 32 as cytokine concentration in pg/ml (mean of triplicates+SD). Thedata showed ex vivo cytokine production by splenocytes from mice boostedwith both analogs, and greater response to L426Nva L433Nle than to S425TL426Nva L433Nle.

Example 47 Ex Vivo Cytotoxicity Against a Human Tumor Cell Line afterPeptide Boost with Analog

HHD transgenic mice (n=4) were immunized with a plasmid, pCTLR2,expressing PRAME₄₂₅₋₄₃₃ by direct inoculation into the inguinal lymphnodes of 25 μg in 25 μl of PBS to each lymph node at day 0, 3, 14 and17. This was followed by two peptide boosts (2.5 μg) at day 28 and 31,with the analog L426Nva L433Nle. One week after the boost, splenocyteswere stimulated ex vivo with the native peptide and tested against⁵¹Cr-labeled human tumor cells (PRAME⁺ 624.38 melanoma cells pretreatedor not with IFN-γ; or negative control 624.38 cells, deficient in HLA-A2expression) at various E:T ratios. The analog L426Nva L433Nle elicitedimmune responses that mediated significant cytotoxicity against humantumor cells expressing A2 (624.38), slightly elevated upon theirpre-treatment with IFNγ. In contrast, no significant activity wasmeasured against A2-624.28 control cells. See FIG. 33.

Example 48 In Vitro Immunization to PRAME₄₂₅₋₄₃₃

In vitro immunization was carried out according to the general schemepresented in FIG. 34. Peripheral blood mononuclear cells (PBMCs) wereobtained from healthy donors (HLA-A*0201⁺) by Ficoll-separation. FreshPBMCs (2.5×10⁶), together with 5 ng/ml PRAME₄₂₅₋₄₃₃ or peptide analogwere plated in T-cell culture medium. Subsequently 20 IU/ml ofinterleukin-2 was added to each well after 72 and 96 hours andadditional peptide (5 ng/ml) was added at day 7. Cultures weremaintained for an additional 10 days before effector cells wereharvested and used in tetramer staining. IVS PBMCs were labeled withPRAME₄₂₅₋₄₃₃ tetramer and analyzed on the FACSCalibur (BD, San Jose,Calif.). Quadrants were set based on negative controls, stained withirrelevant HBV tetramer and SSX2 tetramer, and a minimum of 10,000 gatedevents were captured. Tetramer-positive cells were expressed as apercentage of the lymphocyte population. PRAME₄₂₅₋₄₃₃-specific tetramerswere significantly enhanced following IVS with peptide analog ascompared with native peptide. See FIG. 35. This demonstrates that theanalog can be a preferable immunogen.

Example 49 pCTLR2, a Plasmid Expressing the PRAME425-433 Epitope

pCTLR2 is a recombinant DNA plasmid vaccine encoding one polypeptidewith an HLA A2-specific CTL epitope from PRAME, amino acid residues425-433, SLLQHLIGL (SEQ ID NO. 71), and an epitope cluster region ofPRAME, amino acids 422-509. The cDNA sequence for the polypeptide in theplasmid is under the control of promoter/enhancer sequence fromcytomegalovirus (CMVp), which allows efficient transcription ofmessenger for the polypeptide upon uptake by antigen presenting cells.The bovine growth hormone polyadenylation signal (BGH polyA) at the 3′end of the encoding sequence provides signal for polyadenylation of themessenger to increase its stability as well as translocation out ofnucleus into the cytoplasm. To facilitate plasmid transport into thenucleus, a nuclear import sequence (NIS) from Simian virus 40 wasinserted in the plasmid backbone. One copy of CpG immunostimulatorymotif was engineered into the plasmid to further boost immune responses.Lastly, two prokaryotic genetic elements in the plasmid are responsiblefor amplification in E. coli, the kanamycin resistance gene (Kan R) andthe pMB bacterial origin of replication. (See FIG. 36).

Immunogen Translation Product Sequence

The amino acid sequence of the encoded polypeptide (150 amino acidresidues in length) is given below.

malqsllqhliglsnlthylypvplesyedihgtlhlerlaylharlrellcelgrpsmvwlsanpcphcgdrtfydpepilcpcfmpnkrsllqhliglgdaaysllqhliglispekeeqyiasllqhliglkrpsikrsllqhligl(SEQ ID NO: 196).

The first 89 amino acid residues are an epitope cluster regionrepresenting PRAME 422-509. Within this epitope cluster region, a numberof potential HLA A2-specific CTL epitopes have been found using avariety of epitope prediction algorithms. Amino acid residues 90-150 arean epitope liberation (Synchrotope™) sequence with four copies of PRAME425-433 CTL epitope (boldface) embedded. Flanking the defined PRAME CTLepitope are short amino acid sequences that have been shown to play animportant role in the processing of the PRAME CTL epitope. In addition,the amino acid sequence ispekeeqyia (SEQ ID NO. 197) (corresponding toPRAME amino acid 276-286, in italics) was engineered into thesting-of-beads region to facilitate the detection of expression ofencoded polypeptide.

Using a variety of immunological assays, including tetramer, ELISPOT,ELISA, and cytotoxicity, strong CTL responses specific for epitopePRAME₄₂₅₋₄₃₃ have been detected from HLA-A2 transgenic mice immunizedwith the pCTLR2 plasmid, suggesting immunogenic potency for pCTLR2.These data indicated that the plasmid was taken up by antigen presentingcells, the encoded polypeptide synthesized and proteolytically processedto produce the nonamer epitope peptide, and the nonamer epitope peptideHLA-A2 bound for presentation.

Plasmid Construction

Stepwise ligation of sets of long complementary oligonucleotidesresulted in generation of cDNA encoding amino acid residues in the“String-of-Beads” epitope liberation sequence (amino acids 90-150).These cDNA bore appropriate cohesive ends for restriction enzymes thatcan be used for further ligation with cDNA encoding the PRAME epitopecluster region (amino acid 1-89), which were amplified by performing PCRon cDNA encoding PRAME as template. The entire insert was then ligatedinto vector backbone between Afl II and EcoR I sites. The entire codingsequence was verified by DNA sequencing.

Example 50 Generation of Antigen Specific T Cell Responses

H-2 class I-negative, HLA-A2.1-transgenic HHD mice were housed underpathogen-free conditions and used for evaluation of the immunogenicityof HLA-A2.1-restricted human tumor-associated cytotoxic T lymphocyte(CTL) epitopes. Female mice 8-12 weeks of age were used forintralymphatic immunization and for isolation of splenocytes for in vivocytotoxicity studies. The mice were immunized via bilateral inguinallymph node injection. Mice were anesthetized by inhalation ofisofluorane and surgeries were conducted under aseptic conditions.Following preparation for surgery, an incision 0.5 cm in length was madein the inguinal fold and the inguinal lymph node was exposed. A maximumvolume of 25 μl (25 μg) of plasmid DNA vaccine or peptide was injecteddirectly into the lymph node using a 0.5 mL insulin syringe. The woundwas closed with sterile 6-0 nylon skin sutures.

Example 51 Ex Vivo Cytotoxicity Against Human Tumor Cells

HHD transgenic mice (n=4/group) were immunized with the plasmid pSEM(described more fully in U.S. patent application Ser. No. 10/292,413(Pub. No.20030228634 A1), which is incorporated herein by reference inits entirety) expressing melan-A₂₆₋₃₅ A27L epitope analog by directinoculation into the inguinal lymph nodes with 25 μg in 25 μl ofPBS/each lymph node at day 0, 3, 14 and 17. This was followed by twoadditional peptide boosts (same amount) at day 28 and 31, with theanalogs A27L, A27Nva, or A27L V35Nva. One week after the boost,splenocytes were stimulated ex vivo with the native melan-A₂₆₋₃₅ peptideand tested against ⁵¹Cr-labeled human tumor cells (624.38 cells) atvarious E:T ratios. The resulting immunity after boosting with the A27Lor A27Nva analogs was comparable and more effective than the nativepeptide EAAGIGILTV (SEQ ID NO. 100) (FIG. 37). Because the primingplasmid expresses the A27L analog, the experiment had a potential biasin favor of that peptide. Thus, the substantial cytotoxicity obtainedwith the A27Nva analog may be an underestimate of it potency if primingmade use of that same sequence.

Example 52 Tetramer Analysis

Enumeration of CD8+ antigen-specific T cells requires cognaterecognition of the T cell receptor (TCR) by a Class I MHC/peptidecomplex. This can be done using Class I MHC tetramers which are composedof a complex of four HLA MHC Class I molecules each bound to thespecific peptide and conjugated with a fluorescent protein. Thus,tetramer assays allow quantitation of the total T cell populationspecific for a given peptide complexed in a particular MHC molecule.Furthermore, because binding does not depend on functional pathways,this population includes all specific CD8+ T cells regardless offunctional status. The CTL response in immunized animals was measured byco-staining mononuclear cells isolated from peripheral blood afterdensity centrifugation (Lympholyte Mammal, Cedarlane Labs) withHLA-A*0201 MARTI (ELAGIGILTV (SEQ ID NO. 100))-PE MHC tetramer (BeckmanCoulter, T01008) or a Tyrosinase₃₆₉₋₃₇₇ (YMDGTMSQV (SEQ ID NO. 94))specific tetramer reagent (HLA-A*0201 Tyrosinase-PE, Beckman Coulter)and FITC conjugated rat anti-mouse CD8a (Ly-2) monoclonal antibody (BDBiosciences). Data was collected using a BD FACS Calibur flow cytometerand analysed using cellquest software by gating on the lymphocytepopulation and calculating the percent of tetramer⁺ cells within theCD8⁺ CTL population.

Example 53 Tetramer Staining (Plasmid Priming, Peptide Boost—NativeVersus Analog)

Two groups of HHD transgenic mice (n=8) were immunized with plasmidexpressing Tyrosinase 369-377 by direct inoculation into the inguinallymph nodes with 25 μg in 25 μl of PBS/each lymph node at day 0, 3, 14and 17. This was followed by two additional peptide boosts (similaramount) at day 28 and 31, of natural peptide or the 377Nva analog. Tendays later, the immune response was monitored using a Tyrosinase₃₆₉₋₃₇₇specific tetramer reagent (HLA-A*0201 Tyrosinase-PE, Beckman Coulter).Individual mice were bled via the retro-orbital sinus vein and PBMC wereisolated using density centrifugation (Lympholyte Mammal, CedarlaneLabs) at 2000 rpm for 25 minutes. PBMC were co-stained with a mousespecific antibody to CD8 (BD Biosciences) and the Tyrosinase tetramerreagent and specific percentages were determined by flow cytometeryusing a FACS caliber flow cytometer (BD Biosciences). The percentages ofTyrosinase specific CD8+ cells show that replacement of the nativepeptide with the analog preserved the expansion of Tyrosinase-specificsubset. The trend indicates that the analog can improve on the expansionof Tyrosinase specific T cells (FIG. 38).

Example 54 In Vivo Cytotoxicity and Tetramer Staining (Head to HeadComparison Between Native Peptide and a Panel of Analog Candidates)

Four groups of HHD transgenic mice (n=6) were immunized with plasmid(pSEM) expressing Tyrosinase₃₆₉₋₃₇₇ and Melan-A₂₆₋₃₅ A27L epitopes bydirect inoculation into the inguinal lymph nodes of 25 μg of plasmid in25 μl of PBS per lymph node at day 0, 3, 14 and 17. This was followed bytwo peptide boosts (similar amount) at days 28 and 31, of Melan-A₂₆₋₃₅A27L into the left inguinal lymph node and Tyrosinase₃₆₉₋₃₇₇ analogs,bearing substitutions at the primary and/or secondary anchor residues,into the right lymph node. As controls, mice immunized with plasmid onlyor native mice were used.

To evaluate the in vivo response against natural Tyrosinase and Melan Aepitopes, splenocytes were isolated from littermate control HHD mice andincubated separately with 20 μg/ml of natural peptide (Melan-A₂₆₋₃₅ orTyrosinase₃₆₉₋₃₇₇) for 2 hours in HL-1 serum free medium (Cambrex) at aconcentration of 20×10⁶ cells/mL. These cells were then stained withCFSE (Vybrant CFDA SE cell tracer kit, Molecular Probes) (1 and 2.5 μMrespectively, for 15 minutes) and intravenously co-injected intoimmunized or naive control HHD mice with an equal number of controlnon-peptide coated splenocytes stained with CFSE^(lo) fluorescence (0.4μM). Eighteen hours later the specific elimination of target cells wasmeasured by removing the spleen from challenged animals and measuringCFSE fluorescence in the resulting cell suspensions by flow cytometry.The relative depletion of the populations corresponding topeptide-loaded splenocytes was calculated relative to the control(unloaded) population and expressed as % specific lysis. In addition,the frequency of Tyrosinase₃₆₉₋₃₇₇- and Melan-A₂₆₋₃₅-specific T cells,was evaluated by tetramer/CD8 co-staining (HLA-A*0201-tetramers, BeckmanCoulter).

The tyrosinase analog V377Nva was capable of expanding the population oftyrosinase-specific T cells and amplifying cytotoxic immunity, similarlyto the native peptide and greater than the Tyrosinase analog M370VV377Nva (FIG. 39).

Example 55 Ex Vivo Cytotoxicity Against Human Tumor Cells

HHD transgenic mice (n=4/group) were immunized (according to the generalprotocol in FIG. 40) with plasmid (pSEM) expressing theTyrosinase₃₆₉₋₃₇₇ epitope by direct inoculation into the inguinal lymphnodes of 25 μg of plasmid in 25 μl of PBS per lymph node at day 0, 3, 14and 17. This was followed by two peptide boosts (same amount) at day 28and 31, with the native peptide or analogs bearing substitutions atprimary anchor residues P2 and PΩ (370 and 377). One week after theboost, splenocytes were stimulated ex vivo with the nativeTyrosinase₃₆₉₋₃₇₇ peptide and assayed against ⁵¹Cr-labeled human tumorcells (624.38 cells) at various E:T ratios. Both the native peptide andthe M370V V377Nva analog generated robust cytotoxicity against 624.38cells (FIG. 41). Whereas there was some dilution of cytolytic activitywith the native peptide, there was none with the analog, therebyreinforcing the indication of greater immunogenicity gained from thetetramer results in Example 52. Together with the preceding example,this observation illustrates the usefulness of complementing morestringent assays (in vivo cytotoxicity and tetramer staining) with moresensitive assays (ex vivo cytotoxicity after in vitro stimulation) tooutline potentially useful analogs.

Example 56 Binding and Stability (Half-Life) Characterization of NativePeptides and Analogs by Itopia System

Because the binding and the stability of the peptides bound to the MHC 1complexes are essential to get a good immune response, native epitopesand analogs thereof were tested for their binding and stability(T_(1/2)) to A0201 restricted MHC Class 1 molecules using iTopia Epitopediscovery System (also discussed in Example 31). The binding and offrate(T_(1/2)) was determined for six native epitopes and their analogs whichwere designed to enhance the immune response. The native peptides andanalogs were as follows: NY-ESO-1₁₅₇₋₁₆₅, NY-ESO-1₁₅₇₋₁₆₅ (L158Nva,C165V)), Melan-A₂₆₋₃₅, Melan-A₂₆₋₃₅ (A27Nva), SSX-2₄₁₋₄₉, SSX-2₄₁₋₄₉(A42V), Prame₄₂₅₋₄₃₃, Prame₄₂₅₋₄₃₃ (L426Nva, L433Nle),Tyrosinase₃₆₉₋₃₇₇, Tyrosinase₃₆₉₋₃₇₇ (V377Nva), PSMA₂₈₈₋₂₉₇ andPSMA₂₈₈₋₂₉₇(I297V).

Table-10 (below) shows the average values of peptide binding andstability or half-life of six analogs and their native peptides. Theanalogs of SSX-2₄₁₋₄₉ and NY-ESO-1₁₅₇₋₁₆₅ showed substantial increase inthe percent binding compared to the native peptides. The analogs ofMelan-A₂₆₋₃₅, Psma₂₈₈₋₂₉₇, Prame₄₂₅₋₄₃₃ and Tyrosinase₃₆₉₋₃₇₇ showed amarginal increase in the percentage binding of the peptides compared tothe native peptides.

Melan A₂₆₋₃₅ (A27Nva) showed substantial increase in the stability(half-life) compared to the native peptide. Significant increase in thestability was observed for other analogs Psma₂₈₈₋₂₉₇ (I297V),NY-ESO-1₁₅₇₋₁₆₅ (L158Nva, C165V), Prame₄₂₅₋₄₃₃ (L236Nva, L433Nle) andSSX-2₄₁₋₄₇ (A42V). No significant differences were observed in thehalf-life of the analog Tyrosinase₃₆₉₋₃₇₇ (V377Nva) compared to thenative peptide. The differences observed in the half life of the analogsmay be due to the difference in the mechanism of binding of each analogto the MHC 1 molecules TABLE 10 THE AVERAGE VALUES OF PERCENT BINDINGAND THE STABILITY (HALF-LIFE) OF NATIVE PEPTIDES AND THEIR ANALOGS. %Peptide Half-Life Binding # Peptide Name (T½) at T0 1 Melan-A 26-35 1.8574 2 Melan-A 26-35 (A27Nva) 13.55 79 3 PSMA 288-297 7.34 85 4 PSMA288-297(I297V) 16.72 92 5 Nyeso-1 157-165 12.74 63 6 Nyeso-1 157-165(L158Nva, C165V)) 16.98 100 7 PRAME 425-433 12.42 79 8 Prame 425-433(L426Nva, L433Nle) 16.87 89 9 SSX2 41-49 9.90 55 10 SSX2 41-49 (A42V)15.01 71 11 Tyrosinase 369-377 14.39 73 12 Tyrosinase 369-377 (V377Nva)14.74 77

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods may beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the invention extends beyond the specifically disclosedembodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements of the invention have beendisclosed. Still further variations and alternate elements will beapparent to one of skill in the art. Among these variations, withoutlimitation, are the specific number of antigens in a screening panel ortargeted by a therapeutic product, the type of antigen, the type ofcancer, and the particular antigen(s) specified. Various embodiments ofthe invention can specifically include or exclude any of thesevariations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferents used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) may be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans may employ such variations asappropriate, and the invention may be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed may be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. An isolated peptide consisting essentially of a sequence in which: P0is X, XX, or XXX, wherein X specifies any amino acid or no amino acid;and P1 is S, K, F, Y, T, Orn, or Hse; and P2 is L, V, M, I, Nva, Nle, orAbu; and P3 is L, Nva, Nle or Abu; and P4 is Q; and P5 is H; and P6 isL, Nva, Nle, or Abu; and P7 is I; and P8 is G, A, S, or Sar; and PΩ atP9 is L, V, I, A, Nle, Nva, Abu, or L-NH₂; and PΩ+1 is X, XX, or XXX,wherein X specifies any amino acid or no amino acid; and wherein thesequence is not SLLQHLIGL (SEQ ID NO: 71).
 2. The isolated peptide ofclaim 1 consisting essentially of the sequence: {K, F, Y, T, Orn, orHse}LLQHLIGL (SEQ ID NO: 72); or S{V, M, I, Nva, Nle, or Abu}LQHLIGL(SEQ ID NO: 73); or SL{Nva, Nle or Abu}QHLIGL (SEQ ID NO: 74); orSLLQH{Nva, Nle or Abu}IGL (SEQ ID NO: 75); or SLLQHLI {A, S, or Sar}L(SEQ ID NO: 76); or SLLQHLIG {V, I, A, Nle, Nva, Abu, or L-NH₂} (SEQ IDNO: 77); or {F, Y, T, Orn, or Hse}{Nva, Nle, M, or I}LQHLIGL (SEQ ID NO:78); or S{Nva, Nle, or M}LQHLIG{Nva, Nle, or V} (SEQ ID NO: 79); or {K,F, Y, T, Orn, or Hse}LLQHLIGV (SEQ ID NO: 80); or {F or T}LLQHLIG{Nle}(SEQ ID NO: 81); or {F or T} {Nva or M}LQHLIG{Nle} (SEQ ID NO: 82). 3.The isolated peptide of claim 2 consisting essentially of the sequence:{F, Y, T, Orn, or Hse}LLQHLIGL (SEQ ID NO: 83); or S{Nva, Nle, orM}LQHLIGL (SEQ ID NO: 84); or SLLQHLIG{Nle, Nva, or L-NH₂} (SEQ ID NO:85); or SLLQH{Nva or Abu}IGL (SEQ ID NO: 86); or S{Nva}LQHLIG{Nle} (SEQID NO: 87); or {F or T} {L or Nva}LQHLIG{Nle} (SEQ ID NO: 88).
 4. Theisolated peptide of claim 3 consisting essentially of the sequence: S{Lor Nva}LQHLIG{Nle} (SEQ ID NO: 89); or T{Nva}LQHLIG{Nle} (SEQ ID NO:90).
 5. The isolated peptide of claim 4 consisting essentially of thesequence: S{Nva}LQHLIG{Nle} (SEQ ID NO: 87).
 6. The isolated peptide ofclaim 1, wherein the peptide has affinity for a class I MHC peptidebinding cleft.
 7. The isolated peptide of claim 6, wherein the class IMHC is HLA-A2.
 8. An isolated peptide comprising 1 to 3 substitutions inthe sequence SLLQHLIGL (SEQ ID NO: 71) having an affinity for a class IMHC binding cleft that is similar to or greater than the affinity ofSLLQHLIGL (SEQ ID NO: 71) for said class I MHC binding cleft.
 9. Theisolated peptide of claim 8, wherein the halftime of dissociation issimilar to or greater than the halftime of dissociation of SLLQHLIGL(SEQ ID NO: 71) from said class I MHC binding cleft.
 10. The isolatedpeptide of claim 8, that is recognized by T cells with specificity forthe peptide SLLQHLIGL (SEQ ID NO:71).
 11. A class I MHC/peptide complex,wherein the peptide has the sequence of the peptide of claim
 1. 12. Theclass I MHC/peptide complex of claim 11, that is cross-reactive with aTCR that recognizes a class I MHC/PRAME₄₂₅₋₄₃₃ complex.
 13. The class IMHC/peptide complex of claim 12, wherein the class I MHC/complex is anHLA-A2/PRAME₄₂₅₋₄₃₃ complex.
 14. A polypeptide comprising the peptidesequence of claim 1 embedded within a liberation sequence.
 15. Animmunogenic composition comprising the peptide of claim
 1. 16. Animmunogenic composition comprising the polypeptide of claim
 14. 17. Anucleic acid encoding the polypeptide of claim
 14. 18. A nucleic acidmeans for expressing the peptide of claim
 1. 19. An immunogeniccomposition comprising the nucleic acid of claim 17 or
 18. 20. A methodof inducing, maintaining, or amplifying a CTL response comprisingintranodal administration of the composition of claim
 15. 21. A methodof entraining a class I MHC-restricted T cell response comprisingintranodal administration of the composition of claim 15 and animmunopotentiating agent.
 22. A method of inducing, maintaining, orentraining a CTL response comprising intranodal administration of thecomposition of claim 19.